lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): OC(CN[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1)(c1cccnc1)C(F)(F)F
InChIKey: SQWIAXGMZFHLAX-WBEUYKKISA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 445.45, ALogP 3.07, HBA 7, HBD 3, PSA 104.02, QED 0.44
Compound (SMILES): CC(C)N(C)c1nc2c(c(Nc3ccc(C(F)(F)F)cc3)n1)CCN(c1ncccc1C(F)(F)F)CC2
InChIKey: ZBYHLZHTSDBFEM-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: O35433
Assay: Functional Assay: The assay was performed as described below, using HEK293 cells transfected with rat TRPV1 (rTRPV1/HEK293). These cells had a geneticin selection marker and were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, penicillin/streptomycin (50 units/mL), and 500 μg/mL geneticin in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 524.51, ALogP 6.1, HBA 6, HBD 1, PSA 57.18, QED 0.41
Compound (SMILES): NC(=O)C12CC3CC(C1)C(OC(=O)N1CCC(c4ncc(Cl)cn4)C1)C(C3)C2
InChIKey: DJVOJGHZDVCWOV-UHFFFAOYSA-N
Target: 11-beta-hydroxysteroid dehydrogenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28845
Assay: Scintillation Proximity Assay: Inhibition of microsomal preparations of 11.beta.-HSDl by compounds of the present invention, as described essentially previously (K. Solly, SS Mundt, HJ Zokian, GJ Ding, A. Hermanowski-Vosatka, B. Strulovici, and W. Zheng, High-Throughput Screening of 11-Beta-Hydroxyseroid Dehydrogenase Type 1 in Scintillation Proximity Assay Format. Assay Drug Dev Technol 3 (2005) 377-384) was measured. All reactions were carried out in a clear flexible PET Microbeta plates 96-well at room temperature (PerkinElmer). Assay, substrate solution (50mM HEPES, pH7.4,100mM KCl, 5mM NaCl, 2mM MgCl 2, 2mM NADPH and 160nM [3 H] cortisone (1Ci / mmol)) a 49ul and distribution, and starting from the 0.1mM on to start by combining the semi-log (half-log) multiple of the incremental test compound 1uL in DMSO previously diluted (8 points). After pre-incubation of 10 minutes, incubated human 11-HSD1 overexpress the enzyme containing CHO cells isolated from microsomes solution 50uL.
Measurement: IC50 = 1.91 nM (pChEMBL 8.720000267028809)
Descriptors: MW 404.9, ALogP 2.74, HBA 5, HBD 1, PSA 98.41, QED 0.83
Compound (SMILES): Nc1ncnc(N2CCC(c3nc(-c4cccc(C(F)(F)F)c4)c[nH]3)CC2)c1Br
InChIKey: NNFMZOAUPAFRQG-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: AKT Enzyme Assay: A TTP Mosquito liquid handling instrument is used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components are added to a final volume of 12.5 .mu.l: 0.1 ng/.mu.l His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C). 160 uM ATP (Fluka, 02055) 1 mM DTT (Sigma, D0632) 1 mM MgCl2 (Sigma, M1028) 1 .mu.M substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service. 100 mM HEPES pH 7.5 (Calbiochem, 391338)0.015% Brij-35 (Sigma, B4184)The reaction is incubated for 90 min at 25 C, and then stopped by the addition of 70 .mu.l of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate is read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure -2.3 psi, upstream voltage -500, and downstream voltage -3000.
Measurement: IC50 = 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 467.29, ALogP 4.61, HBA 5, HBD 2, PSA 83.72, QED 0.59
Compound (SMILES): NC(=O)c1cc(-c2ccc(F)cc2)c2ccc(Cc3cncnc3)cc2n1
InChIKey: NKCGHGIOYKKQFX-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Binding affinity to human mGluR2 expressed in cell membrane incubated for 1 hr in presence of tritium labeled [3H]-7-((2,5-dioxopyrrolidin-1-yl)methyl)-4-(4-fluorophenyl)quinoline-2-carboxamide by competitive radioligand binding assay
Measurement: Ki = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 358.38, ALogP 3.52, HBA 4, HBD 1, PSA 81.76, QED 0.61
Compound (SMILES): O=C(CC1([C@H]2COC[C@@H](C3CC3)N2S(=O)(=O)c2ccc(F)cc2)CC1)N1CCN(CCO)CC1
InChIKey: ZISKXVLQDDFGDG-FCHUYYIVSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition of recombinant human CYP3A4 expressed in baculovirus infected insect cells preincubated for 30 mins using fluorescent substrate
Measurement: IC50 = 6900 nM (pChEMBL 5.159999847412109)
Descriptors: MW 495.62, ALogP 1.3, HBA 6, HBD 1, PSA 90.39, QED 0.58
Compound (SMILES): COc1cc2c(cc1OC)C(=O)C(CC1CCN(Cc3ccc(Cl)cc3)CC1)C2
InChIKey: VGQNXCDLTRSTAF-UHFFFAOYSA-N
Target: Cholinesterase (SINGLE PROTEIN), Equus caballus
UniProt: P81908
Assay: Inhibition of equine serum BuChE using s-butyrylthiocholine iodide as substrate preincubated for 5 mins followed by substrate addition measured up to 3 mins by Ellman's method
Measurement: IC50 = 89400 nM (pChEMBL 4.050000190734863)
Descriptors: MW 413.95, ALogP 5.01, HBA 4, HBD 0, PSA 38.77, QED 0.67
Compound (SMILES): O=C(Cc1ccc(Cl)cc1Cl)NC1CCOc2ncccc21
InChIKey: BMKDSFJBRRVNJK-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: In Vitro Assay: Cell line generation was performed in general according to established molecular cloning protocols. Specifically, RNA was extracted from human whole blood using the Qiagen RNeasy kit (Qiagen, CH) according to the manufacturer's instructions. Subsequently cDNA was made (Superscript II, Invitrogen AG, CH) and the human P2X7 gene (genbank ref. BC011913) was amplified with the following primers: ATCGCGGCCGCTCAGTAAGGACTCTTGAAGCCACT and CGCCGCTAGCACCACCATGCCGGCCTGCTGCAGCTGCA. The amplified sequence was subsequently ligated into a pcDNA3.1 (+) NotI, NheI digested plasmid. Human embryonic kidney (HEK) cells (ATCC CRL-1573, Manassas, Va., USA) were transfected with the pcDNA3.1 (+). hP2X7 plasmid using lipofectamine 2000 (Invitrogen AG, CH) according to the manufacturer's instructions. Following a 24 h exposure to DNA, cells were trypsinized and re-seeded at low density in the presence of 250 ug Geneticin. Geneticin resistant cells were then selected during two consecutive rounds of cloning by serial limiting dilution with visual inspection.
Measurement: IC50 = 177 nM (pChEMBL 6.75)
Descriptors: MW 337.21, ALogP 3.57, HBA 3, HBD 1, PSA 51.22, QED 0.93
Compound (SMILES): NC1=N[C@](CF)(c2cc(NC(=O)c3ncc(Cl)cc3F)cc(F)c2F)C[C@@H](C(F)(F)F)O1
InChIKey: HLDJICBVNWASST-YVEFUNNKSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro BACE Cell-Based Assay: The cell-based assay measures inhibition or reduction of Aβ40 in conditioned medium of test compound treated cells expressing amyloid precursor protein. Cells stably expressing Amyloid Precursor Protein (APP) were plated at a density of 40K cells/well in 96 well plates (Costar). The cells were cultivated for 24 hours at 37° C. and 5% CO2 in DMEM supplemented with 10% FBS. The test compounds were then added to cells in 10-point dose response concentrations with the starting concentration being either 100 uM or 10 uM. The compounds were diluted from stock solutions in DMSO and the final DMSO concentration of the test compounds on cells was 0.1%. After 24 h of incubation with the test compounds the supernatant conditioned media was collected and the Aβ 40 levels were determined using a sandwich ELISA.
Measurement: IC50 = 78 nM (pChEMBL 7.110000133514404)
Descriptors: MW 484.76, ALogP 4.24, HBA 5, HBD 2, PSA 89.6, QED 0.64
Compound (SMILES): COc1cccc(-c2c(/C=C/c3ccc4c(C(=O)NS(C)(=O)=O)cccc4n3)nc3c(N4CCOCC4)ccnn23)c1
InChIKey: UHZZSJCMZUSRKZ-ZRDIBKRKSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Assay: Human recombinant PDE10A2 was expressed in Sf9 cells, using a recombinant baculovirus construct containing the full length sequence containing a 6Ã¿His sequence following the start Met to allow metal affinity purification of the recombinant protein. Cells were harvested and the phosphodiesterase protein was purified by metal chelate chromatography on Ni-sepharose 6FF.The affinity of the compounds of Formula (I) for phophodiesterases (PDE) was measured by a scintillation proximity assay (SPA). PDE Yttrium Silicate SPA beads allow PDE activity to be measured by direct binding of the primary phosphate groups of non-cyclic AMP or GMP to the beads via a complex iron chelation mechanism. The amount of bound tritiated product ([3H]-AMP) is measured by liquid scintillation counting.The compounds were dissolved and diluted in 100% DMSO in polystyrene plates to a concentration of 100-fold the final concentration in the assay. Human PDE10A enzyme solution (10 uL) was added.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 584.66, ALogP 3.65, HBA 10, HBD 1, PSA 128.02, QED 0.3
Compound (SMILES): CNC(=O)c1cc(Oc2ccc(C(=O)/C=C/c3ccccc3)cc2)ccn1
InChIKey: XOPLZHOJEWTTRO-KPKJPENVSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of BRAF (unknown origin) using FAM-labeled peptide as substrate after 1 hr by Lanthascreen assay
Measurement: IC50 = 2400 nM (pChEMBL 5.619999885559082)
Descriptors: MW 358.4, ALogP 4.13, HBA 4, HBD 1, PSA 68.29, QED 0.53
Compound (SMILES): COc1ccc(C(=O)n2c(-c3ccc(OC)cc3)nc3ccccc32)cc1
InChIKey: PXJXUUDEYSHZAX-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Displacement of [3H]CP-55940 from human recombinant CB1 receptor expressed in HEK293 cell membranes incubated for 1.5 hrs
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 358.4, ALogP 4.41, HBA 5, HBD 0, PSA 53.35, QED 0.54
Compound (SMILES): CN(c1ccc(C#N)nn1)[C@@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: INNQJNLPVHXGCK-GFCCVEGCSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 (300/100/40/100M, 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 = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 320.36, ALogP 1.33, HBA 7, HBD 1, PSA 97.62, QED 0.78
Compound (SMILES): O=C(CCc1nc2cccnc2n1Cc1ccc(F)cc1)N[C@@H](CO)c1ccc(Cl)cc1
InChIKey: FYLJXUPWLMKZQB-NRFANRHFSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Inhibition of human recombinant ATX using Rac-1-Palmitoyl-glycero-3-phosphocholine as substrate incubated for 2 hrs using ADHP fluorogenic peroxidase substrate by horseradish peroxidase/choline oxidase-coupled assay
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 452.92, ALogP 4.05, HBA 5, HBD 2, PSA 80.04, QED 0.42
Compound (SMILES): Cc1cc(C(=O)O)c2c(C)nn(Cc3ccc(Oc4ccccc4)cc3)c2n1
InChIKey: MYMWHEACODUIMJ-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 homolog (SINGLE PROTEIN), Mus musculus
UniProt: P97287
Assay: Fluorescence Polarization-Based Binding Assay: Sensitive and quantitative FP-based binding assays were developed and optimized to determine the binding affinities of small-molecule inhibitors to the recombinant Mcl-1, A1/Bfl-1, Bcl-w, Bcl-2, and Bcl-xL proteins. The concentrations of the proteins used in the competitive binding experiments were 10 nM for Mcl-1, 80 nM for Bcl-xL, and 60 nM for Bcl-2. The fluorescent probes, Flu-BID or FAM-BID were fixed at 2 nM for all assays, which binds with Kd values of 2.3 nM, 14.2 nM and 24.1 nM against Mcl-1, Bcl-2 and Bcl-xL respectively. 5 μL of the tested compound in DMSO and 120 μL of protein/probe complex in the assay buffer (20 mM phosphate pH 7.4, 50 mM NaCl, 1 mM EDTA. 0.05% Pluronic F68)s) were added to 96 well black assay plates, incubated at room temperature for 3 h and the polarization values (mP) were measured at an excitation wavelength at 485 nm and an emission wavelength at 530 nm using the plate reader Synergy H1 Hybrid, BioTek.
Measurement: IC50 = 17260 nM (pChEMBL 4.760000228881836)
Descriptors: MW 373.41, ALogP 4.59, HBA 5, HBD 1, PSA 77.24, QED 0.55
Compound (SMILES): COc1ccc(-c2cc(C(N)=O)nc3cc(C(C)Cc4ccc(C)nc4)ccc23)cc1
InChIKey: HKNQVFASPZAUHK-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr- cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 411.51, ALogP 5.06, HBA 4, HBD 1, PSA 78.1, QED 0.48
Compound (SMILES): Fc1ccc(-c2cn(-c3ccc([C@@H]4CNCCO4)cc3)nn2)cc1
InChIKey: LDTOYTSMHSLKNJ-SFHVURJKSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing mouse TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000xg for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized.
Measurement: Ki = 32.2 nM (pChEMBL 7.489999771118164)
Descriptors: MW 324.36, ALogP 2.73, HBA 5, HBD 1, PSA 51.97, QED 0.8
Compound (SMILES): CC1(C(=O)Nc2cc(Oc3ccc(NC(=O)Nc4ccc(Cl)c(C(F)(F)F)c4)cc3F)ccn2)CC1
InChIKey: ZDWVEWBBYMWVIZ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition Assay: [Experimental method]: Use ADP-GLO™ assay kit from Promega to evaluate the inhibitory activity of compounds at B-Raf. [Instrument]: PerKinElmer's ENVISION plate reader. [Material]: Optiplate-384 well plate (PerKinElmer), kinase buffer (50 mM Hepes pH7.5, 1 mM EGTA, 2 mM DTT, 10 mM Mg2+, 0.05% BSA), B-Raf kinase (Millipore, catalogue #14-530-K), GST-MEK1 substrate (Carna, catalogue #07-141-10), Super pure ATP (Promega), ADP-GLO™ assay kit (Promega, catalogue # V9102), DMSO (Sigma, catalogue #34869), purified water (Millipore, type: Milli-Q). [Study conditions]: Mix B-Raf kinase (final concentration: 5 nM) and compound (DMSO final concentration: 0.25%) and preincubate at 30° C. for 20 min, then add ATP (final concentration: 6 μM) and reaction substrate (final concentration: 30 nM), React for 2 hrs at 30° C. After reaction, add ADP-GLO™ reagent, react for 40 min at room temperature, then add ADP-GLO™ detecting agent, react for 30 min at room temperature. After that, use ENVSION to measure the fluorescence.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 522.89, ALogP 7.07, HBA 4, HBD 3, PSA 92.35, QED 0.3
Compound (SMILES): C=CC(=O)Nc1ccc(NS(=O)(=O)c2ccc(Nc3ncc4c(n3)N(C)c3ccc(C)cc3C(=O)N4C)cc2)cc1
InChIKey: HTVKDECMTMXAPH-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: In Vitro Kinase Assay: The enzymatic activities against PI3K-α, PI3K-β, PI3K-γ and PI3K-δ were tested in ADAPTA assays. Activity against AURKB and AURKB were tested in Z′-Lyte assays. All assays were performed with ATP concentrations of Km for each kinase. BRD4_1 binding was tested using an AlphaScreen assay. All protocols are available from Life Technologies.
Measurement: IC50 = 306 nM (pChEMBL 6.510000228881836)
Descriptors: MW 569.65, ALogP 4.81, HBA 8, HBD 3, PSA 136.63, QED 0.27
Compound (SMILES): CN1Cc2c(Cl)cc(Cl)cc2[C@H](c2cccc(S(=O)(=O)NCCOCCOCCNC(=O)CCC(CCC(=O)NCCOCCOCCNS(=O)(=O)c3cccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)(CCC(=O)NCCOCCOCCNS(=O)(=O)c3cccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)NC(=O)CCCC(=O)O)c2)C1
InChIKey: ULEZDZVFJXMHLF-AZOUZOSFSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Inhibition of human NHE3-mediated sodium-dependant hydrogen ion antiport in Opossum kidney cells preincubated in NH4Cl-HEPES buffer containing BCECF-AM dye followed by washout with ammonium free HEPES and subsequent addition of compound in presence of NHE1 antagonist EIPA by fluorescence assay
Measurement: IC50 = 50.12 nM (pChEMBL 7.300000190734863)
Descriptors: MW 1814.69
Compound (SMILES): CN1CCC(=CCn2cc(NC(=O)c3cnn4cccnc34)c(-c3cc(Cl)ccc3OC(F)F)n2)CC1
InChIKey: PSQFWQPEKQYJCE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 2.09 nM (pChEMBL 8.680000305175781)
Descriptors: MW 527.96, ALogP 4.75, HBA 8, HBD 1, PSA 89.58, QED 0.35
Compound (SMILES): CC1(C)C(=O)N(c2ccc(C#N)c(C(F)(F)F)c2)C(=S)N1c1ccc(Cn2ccc3cc(/C=C/C(=O)NO)ccc32)c(F)c1
InChIKey: RPYILBLFWKTSJT-ONNFQVAWSA-N
Target: Androgen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P10275
Assay: Displacement of [3H]mibolerone from androgen receptor (unknown origin) in membranes after 24 hrs by HAP adsorption assay
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 621.62, ALogP 6.15, HBA 6, HBD 2, PSA 101.6, QED 0.09
Compound (SMILES): COc1cc(OCCN)cc(N(CC2CC2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: HWPCUDFQBWGXQP-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 444.54, ALogP 3.92, HBA 8, HBD 1, PSA 91.32, QED 0.42
Compound (SMILES): c1ccc(-c2ccnc(NC3=NC[C@@]4(CN5CCC4CC5)O3)c2)cc1
InChIKey: NWBIEXCBVDJYDQ-FQEVSTJZSA-N
Target: 5-hydroxytryptamine receptor 3A (SINGLE PROTEIN), Homo sapiens
UniProt: P46098
Assay: Antagonist activity at human 5-HT3A expressed in HEK293 cells assessed as reduction in acetylcholine-induced activity preincubated for 30 mins followed by addition of acetylcholine measured for 2 mins by Fluo-4AM dye based FLIPR assay
Measurement: IC50 = 85 nM (pChEMBL 7.070000171661377)
Descriptors: MW 334.42, ALogP 3.01, HBA 5, HBD 1, PSA 49.75, QED 0.92
Compound (SMILES): CCO[C@H]1CC[C@]2(CC1)Cc1ccc(-c3cncc(C(F)(F)F)c3)cc1C21N=C(C)C(N)=N1
InChIKey: ROMALOVSXMWQTO-MOOVDDBPSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: sAPPbeta Release Assay: SH-SY5Y cells were cultured in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids and cryopreserved and stored at −140° C. at a concentration of 7.5-9.5×106 cells per vial. Thaw cells and seed at a conc. of around 10000 cells/well in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids to a 384-well tissue culture treated plate, 100 μL cell susp/well. The cell plates were then incubated for 7-24 h at 37° C., 5% CO2. The cell medium was removed, followed by addition of 30 μL compound diluted in DMEM/F-12 with Glutamax, 10% FCS, 1% non-essential amino acids and 1% PeSt to a final conc. of 1% DMSO. The compounds were incubated with the cells for 17 h (overnight) at 37° C., 5% CO2. Meso Scale Discovery (MSD) plates were used for the detection of sAPPβ release. MSD sAPPβ plates were blocked in 1% BSA in Tris wash buffer (40 μL/well) for 1 h on shake at r.t. and washed 1 time in Tris wash buffer (40 μL/well). 20 μL of medium was transferred to the pre-blocked and washed MSD sAPPβ microplates, and the cell plates were further used in an ATP assay to measure cytotoxicity. The MSD plates were incubated with shaking at r.t. for 2 h and the media discarded. 10 μL detection antibody was added (1 nM) per well followed by incubation with shaking at r.t. for 2 h and then discarded. 40 μL Read Buffer was added per well and the plates were read in a SECTOR Imager.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 456.51, ALogP 5.27, HBA 5, HBD 1, PSA 72.86, QED 0.68
Compound (SMILES): CN(C)CC(C)(C)CNS(=O)(=O)c1ccc(Nc2ccc(Cl)cc2Cl)cc1C(F)(F)F
InChIKey: WEPQNBYDVLRSOG-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: IC50 = 6000 nM (pChEMBL 5.21999979019165)
Descriptors: MW 498.4, ALogP 5.62, HBA 4, HBD 2, PSA 61.44, QED 0.5
Compound (SMILES): Cc1nc2cccc(-c3cc4c([nH]3)CCNC4=O)c2nc1N[C@H]1C[C@@H](NCC(F)F)C1
InChIKey: YRKZFOAKGJGXCI-BETUJISGSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 384-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 426.47, ALogP 3.02, HBA 5, HBD 4, PSA 94.73, QED 0.49
Compound (SMILES): C[C@H](n1cnnc1-c1nc(NC(=O)c2cc(-n3cnc(C4CC4)c3)c(N3CCN4C(=O)OCC4C3)cn2)cs1)C(F)(F)F
InChIKey: FUIPXUTXHGPFCR-LBAUFKAWSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: Time-Resolved Fluorescence Resonance energy transfer (TR-FRET) Immunoassay: The assay measures the phosphorylation level of a biotinylated peptide substrate by the ASK1 kinase using HTRF detection. This is a competitive, time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay, based on HTRF KinEASE-STK manual from Cisbio. Test compound, 1 uM STK3 peptide substrate, 4 nM of ASK1 kinase were incubated with 10 mM MOP buffer, pH. 7.0 containing 10 mM Mg-acetate, 0.025% NP-40, 1 mM DTT, 0.05% BSA and 1.5% glycerol for 30 minutes then 100 uM ATP was added to start the kinase reaction and incubated for 3 hr. Peptide antibody labeled with 1xEu3+ Cryptate buffer containing 10 mM EDTA and 125 nM Streptavidin XL665 were added to stop the reaction and phosphorylated peptide substrate was detected using Envision 2103 Multilabeled reader from PerkinElmer. The fluorescence was measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm was calculated for each well.
Measurement: IC50 = 2.3 nM (pChEMBL 8.640000343322754)
Descriptors: MW 614.61, ALogP 3.88, HBA 12, HBD 1, PSA 136.19, QED 0.33
Compound (SMILES): O=C(Nc1nc2ccccc2s1)c1cccc2c1CN(c1ccc(-c3cnn(CC4CCCCC4)c3C3CC3)c(C(=O)O)n1)CC2
InChIKey: TVJXTXUMENZRDB-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: FL5.12 Cellular Assay: The efficacy of the compounds of Formula (I) can also be determined in cell-based killing assays using a variety of cell lines and mouse tumor models. For example, their activity on cell viability can be assessed on a panel of cultured tumorigenic and non-tumorigenic cell lines, as well as primary mouse or human cell populations. In one exemplary set of conditions, mouse FL5.12 cells transfected with Bcl-XL were cultured under standard conditions in RPMI with 2 mM glutamine, 1% 100 mM sodium pyruvate, 2% 1 M HEPES, 4 uL/L of beta;-mercaptoethanol, 1% penicillin-streptomycin, 10% FBS, and 10% WEHI-3B conditioned media (for IL-3). For assaying the compound activity, the cells were exchanged into an IL-3-depleted deprivation media, which was identical to the growth media except for the absence of FBS and WEHI-3B conditional media, for 2 days. Then the cells were exchanged to 3% FBS assay media (RPMI with 2 mM glutamine, 1% 100 mM sodium pyruvate, 2% 1 M HEPES, 4 uL/L of beta;-mercaptoethanol, 1% penicillin-streptomycin, 10% FBS, and 10% WEHI-3B conditioned media (for IL-3).
Measurement: EC50 = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 632.79, ALogP 7.53, HBA 8, HBD 2, PSA 113.24, QED 0.18
Compound (SMILES): Cc1cc(SCCc2nn(-c3ccc(C(F)(F)F)cc3)nc2C)ccc1OCC(=O)O
InChIKey: DZUFIFNEKRHWLT-UHFFFAOYSA-N
Target: Peroxisome proliferator-activated receptor delta (SINGLE PROTEIN), Mus musculus
UniProt: P35396
Assay: Transactivation of yeast GAL4 DNA binding domain-tagged mouse PPARdelta expressed in HEK293T cells incubated for 20 hrs by luciferase reporter gene assay
Measurement: EC50 <= 10000 nM (pChEMBL 5)
Descriptors: MW 451.47, ALogP 4.7, HBA 6, HBD 1, PSA 77.24, QED 0.5
Compound (SMILES): CC(C)Oc1ccc(S(=O)(=O)N2CCN(c3cc(F)c(F)c(OCC(=O)O)c3)CC2)cc1
InChIKey: BJJRJUGQJSTREC-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q13258
Assay: Displacement of [3H]-PGD2 from PGD2 receptor in human platelet membranes after 2 hrs by micro beta scintillation counting analysis
Measurement: Ki = 870 nM (pChEMBL 6.059999942779541)
Descriptors: MW 470.49, ALogP 2.73, HBA 6, HBD 1, PSA 96.38, QED 0.63
Compound (SMILES): COc1cccc(-c2cnc(N)nc2-c2ccccc2)c1
InChIKey: CHYKMYJLTKUFAB-UHFFFAOYSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Displacement of [3H]DPCPX from recombinant human adenosine A1 receptor expressed in CHO cell membranes after 1 hr by beta scintillation counting method
Measurement: Ki = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 277.33, ALogP 3.4, HBA 4, HBD 1, PSA 61.03, QED 0.8
Compound (SMILES): COc1cc(Nc2nc(C)cc(Nc3cc(C4CC4)[nH]n3)n2)cc(OC)c1
InChIKey: QOSPVZIEZFHAPZ-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) tri-phosphorylated enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 >= 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 366.43, ALogP 3.89, HBA 7, HBD 3, PSA 96.98, QED 0.58
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CN(C(=O)c2ccc(C(=O)N3C[C@H](NC(=O)[C@H](C)NC)C(=O)N(Cc4c(OC)ccc5cc(Br)ccc45)c4ccc(C#N)cc43)cc2)c2cc(C#N)ccc2N(Cc2c(OC)ccc3cc(Br)ccc23)C1=O
InChIKey: HTOSWKJYDYFOEH-KUFAWANDSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature.
Measurement: IC50 = 4495 nM (pChEMBL 5.349999904632568)
Descriptors: MW 1202.96
Compound (SMILES): CCc1ccccc1NC(=O)Nc1ccc(Cl)c(S(=O)(=O)C2CCCNC2)c1O
InChIKey: MKXPBYCTEVMLQI-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25025
Assay: Displacement of [125I]IL8 from recombinant human CXCR2 expressed in CHO cell membranes measured after 1 hr by TopCount scintillation counting method
Measurement: IC50 < 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 437.95, ALogP 3.78, HBA 5, HBD 4, PSA 107.53, QED 0.53
Compound (SMILES): Cc1nc(OC[C@@H]2CN(C(=O)OC(C)(C)C)CCO2)sc1S(=O)(=O)NC(=O)c1ccc(N2CCN(CC3=C(c4ccc(Cl)cc4)CC(C)(C)CC3)CC2)cc1Oc1cnc2[nH]ccc2c1
InChIKey: XAAJAAJGJJIWEJ-QNGWXLTQSA-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. 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 Inhibition constants (Ki) are shown in TABLE 2 below and were determined using Wang's equation (Wang Z.-X. An Exact Mathematical Expression For Describing Competitive Binding Of Two Different Ligands To A Protein Molecule. FEBS Lett. 1995, 360:111-4).
Measurement: Ki = 46.5 nM (pChEMBL 7.329999923706055)
Descriptors: MW 946.59, ALogP 8.69, HBA 13, HBD 2, PSA 168.52, QED 0.12
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(C#N)cc1C21N=C(N)N(CC2CCCO2)C1=O
InChIKey: UPHWMYMEWKSHEJ-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: hERG-Channel Assay: Cells were superfused with a bath solution containing (mM): NaCl (137), KCl (4.0), MgCl2 (1.0), CaCl2 (1.8), Glucose (10), HEPES (10), pH 7.4 with NaOH. Patch pipettes were made from borosilicate glass tubing using a horizontal puller and filled with pipette solution containing (mM): K-aspartate (130), MgCl2 (5.0), EGTA (5.0), K2ATP (4.0), HEPES (10.0), pH 7.2 with KOH. Resistance of the microelectrodes was in the range between 2 and 5 MΩ. Stimulation and Recording:Membrane currents were recorded using an EPC-10 patch clamp amplifier and PatchMaster software. hERG-mediated membrane currents were recorded at 35° C., using the whole-cell configuration of the patch-clamp technique. Transfected HEK293 cells were clamped at a holding potential of -60 mV and hERG-mediated inactivating tail currents were elicited using a pulse pattern with fixed amplitudes (activation/inactivation: 40 mV for 2000 ms; recovery: -120 mV for 2 ms; ramp to 40 mV in 2 ms; inactivating tail current: 40 mV for 50 ms) repeated at 15 s intervals. During each inter-pulse interval 4 pulses scaled down by a factor of 0.2 were recorded for a P/n leak subtraction procedure. R. compensation was employed up to a level that safely allowed recording devoid of ringing.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 408.5, ALogP 2.22, HBA 6, HBD 1, PSA 100.94, QED 0.83
Compound (SMILES): O=c1[nH]c(=O)n(-c2cccc(C(F)(F)F)c2)c2c1cnc1ccc(-c3cnc4[nH]ccc4c3)nc12
InChIKey: VVVPWJKRDCCYGB-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: mTOR Enzymatic Assay: Agents: 1-fold kinase buffer: 50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 3 mM MnCl2, 0.01% Tween-20, 2 mM DTT. 4-fold kinase solution: mTOR kinase was added to 1-fold kinase buffer to form 4-fold kinase solution with a final concentration of 2.5 nM. 2-fold substrate and ATP solution: the substrate 4EBP1 and ATP were added to 1-fold kinase buffer to form 2-fold substrate solution with final concentration of 50 nM of 4EBP1 and 10.8 μM of ATP. 4-fold test substance solutions: 100-fold test substance solutions in different gradient concentrations were formulated with 100% DMSO, and diluted by 25 folds with 1-fold kinase buffer to form 4-fold test substance solutions in different gradient concentrations. Detection solution: a detection solution containing 2-fold final concentrations of EDTA and 4EBP1 phosphorylated antibody was formulated, wherein the final concentration for EDTA was 8 mM, and the final concentration for the 4EBP phosphorylated antibody was 2 nM. Procedure: To each well of a 384-well plate was added 2.5 μL of the 4-fold test substance solutions in gradient concentrations. The replication was made. To each well was added 2.5 μL of 4-fold kinase solution, and then the plate was incubated for 10 mins. Then to each well was added 5 μL/of 2-fold substrate and ATP solution, and then the plate was incubated at room temperature for 1 hour. Finally, 10 μL of the detection solution was added to terminate the reaction. After 60 mins, the data Lance signal (665 nM) was read from Envision.
Measurement: IC50 = 1.12 nM (pChEMBL 8.949999809265137)
Descriptors: MW 474.4, ALogP 4.18, HBA 6, HBD 2, PSA 109.32, QED 0.37
Compound (SMILES): c1cc2[nH]cc(-c3cncc(C4CC4)n3)c2cc1-c1nnc(N2CCNCC2)o1
InChIKey: JTESMRSKGHICMR-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 21.53 nM (pChEMBL 7.670000076293945)
Descriptors: MW 387.45, ALogP 2.96, HBA 7, HBD 2, PSA 95.76, QED 0.56
Compound (SMILES): CCCCN(CCC(=O)NCCN1CCN(C)CC1)C(=O)c1nn(-c2ccc(C(=O)NS(=O)(=O)c3ccc4c(c3)CCN4Cc3ccc(Cl)c(Cl)c3)cc2C(=O)N2CCc3ccccc3C2)c(C)c1Cl
InChIKey: OMVAQZKUAGATGJ-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Fluorescence Resonance Energy Transfer Assay (FRET): The assays were performed in black flat-bottom 384-well plates. The final assay volume was 55 μl prepared from additions of Biotin-Bcl-xL (Bcl-xL: GENBANK® Accession No. Q07817), fluoresceinated 18-mer BIM peptide (NH2-YYANFEDGIRRLEQAIWI-[FAM]) (SEQ ID NO: 1), Streptavidin-Terbium FRET detection reagent, and test compounds in assay buffer consisting of 20 mM Sodium Phosphate, 1 mM EDTA, 50 mM NaCl, and 0.05% Pluronic Acid. The reaction was incubated at room temperature for 60 minutes. After 60 minutes, 5 μL of Streptavidin-Terbium FRET detection reagent (Perkin Elmer) was added to the reaction mixture and incubated at room temperature in the dark for 30 mins. The FRET signal generated by the reaction was detected on the Envision Plate Reader Inhibition data were calculated from FRET values generated by the no protein control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay was 10 nM Bcl-xL, 5 nM fluo
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 1031.51
Compound (SMILES): COc1cc(Nc2nc(Cl)cc3c(C(C)C)n[nH]c23)cc(OC)c1OC
InChIKey: FUEDGAAZRJTKOK-UHFFFAOYSA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
Assay: Binding affinity to wild type A3R (unknown origin) expressed in Flp-In-CHO cells assessed as dissociation constant by by Schild analysis
Measurement: Kd = 549.54 nM (pChEMBL 6.260000228881836)
Descriptors: MW 376.84, ALogP 4.5, HBA 6, HBD 2, PSA 81.29, QED 0.62
Compound (SMILES): CCC1(CC(C(=O)Nc2ccc(-c3noc(=O)[nH]3)cc2)n2cc(OC)c(-c3cc(Cl)ccc3C#N)cc2=O)COC1
InChIKey: GNLXHDHFJKLSJD-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibition Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM Tris/HCl pH 7.4; 100 mM sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of plasma kallikrein from Kordia (0.6 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the substrate H-Pro-Phe-Arg-AMC dissolved in assay buffer (10 μl in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 9.4 nM (pChEMBL 8.029999732971191)
Descriptors: MW 576.01, ALogP 4.39, HBA 9, HBD 2, PSA 152.24, QED 0.3
Compound (SMILES): CS(=O)(=O)N1CCN(Cc2csc3c(-c4cncnc4)nc(N4CCOCC4)nc23)CC1
InChIKey: VFDXWHVZENWDNL-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Enzymatic Inhibitory Activity Assay:   1. Buffer preparation: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS.  2. Compound was formulated in 100% DMSO in a concentration gradient, deposited to a 384-well plate to make final DMSO concentration of 1%.  3. PI3Kα and PI3Kδ enzymes were diluted to be the optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT. Transferred to a 384-well plate and incubated with the compound for a certain time.  4. Substrate was diluted to an optimum concentration with following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT, 50 μM PIP2, Km ATP. The reaction was performed in a 384-well plate for 1 h at room temperature for PI3Kα and 2 hrs at room temperature for PI3Kδ.  5. Read the conversion rate using Caliper Reader, and calculate the inhibition rate as the average of two tests.
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 475.6, ALogP 1.06, HBA 10, HBD 0, PSA 104.65, QED 0.54