lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Oc1cc(O)c(-c2ccnn2-c2ccccc2F)cc1I
InChIKey: XGMCBPGHJJKCJC-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Recombinant Hsp90 [His-Tev-huHsp90-alpha (9-236)] was immobilized on a Biacore CM5 chip at 25 degrees C and a flow rate of 30 uL/min using amine coupling at pH 4.50 according to Biacore standard protocol. Hsp90 was applied at a concentration of 20 ug/mL with 50uM 17-DMAG.Data sets were processed and analyzed using the software Biacore 4000 Evaluation. Solvent corrected and double-referenced association and dissociation phase data were fitted to a simple 1:1 interaction model with mass transport limitations
Measurement: Kd = 1662.67 nM (pChEMBL 5.78000020980835)
Descriptors: MW 396.16, ALogP 3.69, HBA 4, HBD 2, PSA 58.28, QED 0.65
Compound (SMILES): CCCNC(=O)N1CC(CC#N)(n2cc(C(N)=O)c(Nc3ccccc3)n2)C1
InChIKey: QYGXGZKTASIATK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 106 nM (pChEMBL 6.96999979019165)
Descriptors: MW 381.44, ALogP 1.77, HBA 6, HBD 3, PSA 129.07, QED 0.67
Compound (SMILES): CC(C)n1cnnc1-c1cccc(NC(=O)c2ccccc2)n1
InChIKey: DYNFVAGQQVHYSC-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: Inhibition of human ASK1 using myelin basic protein substrate preincubated for 20 mins before [33P]-ATP addition and measured after 2 hrs by filter-binding method
Measurement: IC50 = 607 nM (pChEMBL 6.21999979019165)
Descriptors: MW 307.36, ALogP 3.17, HBA 5, HBD 1, PSA 72.7, QED 0.8
Compound (SMILES): O=C(NC1CCC(O)CC1)c1cnn2ccc(NC3CCc4ccccc43)nc12
InChIKey: IJRFYMDEHNCGGT-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK1 and TYK2 Inhibition Assay: To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 uL kinase reactions containing 1.5 nM JAK1, 0.2 nM purified JAK2 or 1 nM purified TYK2 enzyme, 100 mM Hepes pH7.2, 0.015% Brij-35, 1.5 uM peptide substrate, 25 uM ATP, 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22Â° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 uL of an EDTA containing solution (100 mM Hepes 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. Ki values were then determined using the Morrison tight binding model (Morrison, J. F., Biochim. Biophys. Acta. 185:269-296 (1969).
Measurement: Ki = 50.9 nM (pChEMBL 7.289999961853027)
Descriptors: MW 391.48, ALogP 2.86, HBA 6, HBD 3, PSA 91.55, QED 0.64
Compound (SMILES): O=C(c1ccc2[nH]ccc2c1)N1CCN(C2c3ccccc3-c3ccc([N+](=O)[O-])cc32)CC1
InChIKey: VGMFNTBFKQVZBP-UHFFFAOYSA-N
Target: Enoyl-[acyl-carrier-protein] reductase [NADH] (SINGLE PROTEIN), Mycobacterium tuberculosis
UniProt: P9WGR1
Assay: Inhibition of Mycobacterium tuberculosis InhA using octenoyl-CoA as substrate in presence of NADH and measured for 10 mins by colorimetric assay
Measurement: IC50 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 438.49, ALogP 4.6, HBA 4, HBD 1, PSA 82.48, QED 0.37
Compound (SMILES): O=C(CN1c2ccccc2Sc2ccccc21)NNC(=S)NCc1ccccc1
InChIKey: IXLCNZKPZUWIEO-UHFFFAOYSA-N
Target: Peroxisome proliferator-activated receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P37231
Assay: Displacement of Fluoromone from GST-tagged recombinant human PPARgamma ligand binding domain by LanthaScreen TR-FRET assay
Measurement: EC50 = 2800 nM (pChEMBL 5.550000190734863)
Descriptors: MW 420.56, ALogP 3.98, HBA 4, HBD 3, PSA 56.4, QED 0.44
Compound (SMILES): COC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3c3cc(Br)ccc3F)ccc2[nH]1
InChIKey: PDNIIKHCAFQJSY-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 511.31, ALogP 4.5, HBA 5, HBD 3, PSA 107.55, QED 0.38
Compound (SMILES): N#Cc1ccc(CN2CCC(c3ncn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: QSKZDSFBRGYQHC-UHFFFAOYSA-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 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 356.43, ALogP 3.46, HBA 5, HBD 1, PSA 73.01, QED 0.61
Compound (SMILES): CCC(=O)Nc1cc2c(Nc3ccc4c(c3)CCN4Cc3ccccn3)ncnc2cc1OC
InChIKey: RSJGJVCLGCMCLO-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Irreversible inhibition of GST-tagged ERBB2 (unknown origin) (Ile-675 to Val-1256 residues) expressed in baculovirus infected Sf9 insect cells assessed as reduction in Glu/Tyr copolymer phosphorylation after 6 mins by ELISA
Measurement: IC50 = 897 nM (pChEMBL 6.050000190734863)
Descriptors: MW 454.53, ALogP 4.69, HBA 7, HBD 2, PSA 92.27, QED 0.42
Compound (SMILES): CC(C)(O)[C@H](CC1CC1)NC(=O)c1ccc(C2CC2)c(OCC2CC2)n1
InChIKey: ITZSSVBCGHINKH-SFHVURJKSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 358.48, ALogP 3.42, HBA 4, HBD 2, PSA 71.45, QED 0.71
Compound (SMILES): COc1cccc(-c2cccc(OCC(O)CN3CCc4ccccc4C3)c2)c1
InChIKey: VMSAJLLFQIHJIZ-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Inhibition of PRMT5 in human Z138 cells assessed as decrease in di-methyl arginine levels after 96 hrs by Western blot method
Measurement: EC50 = 1001 nM (pChEMBL 6)
Descriptors: MW 389.5, ALogP 4.16, HBA 4, HBD 1, PSA 41.93, QED 0.66
Compound (SMILES): Oc1ccccc1CCCNCc1ccccc1F
InChIKey: YRIHGAMJWFKQJD-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Inhibition of human SERT expressed in HEK293 cells assessed as reduction in [3]5-HT uptake
Measurement: Ki = 14000 nM (pChEMBL 4.849999904632568)
Descriptors: MW 259.32, ALogP 3.25, HBA 2, HBD 2, PSA 32.26, QED 0.78
Compound (SMILES): O=C(O)c1ccc(NC(=O)C(Cc2ccccn2)n2ccc(-c3cc(Cl)ccc3C(F)(F)F)cc2=O)cc1
InChIKey: CKFCLGOBAUZLCA-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 = 780 nM (pChEMBL 6.110000133514404)
Descriptors: MW 541.91, ALogP 5.7, HBA 5, HBD 2, PSA 101.29, QED 0.31
Compound (SMILES): Cc1cccc2cc([C@H](C)Nc3ncnc4ccccc34)n(-c3ccccc3)c(=O)c12
InChIKey: RULMFBNFMMHRPM-SFHVURJKSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 406.49, ALogP 5.42, HBA 5, HBD 1, PSA 59.81, QED 0.43
Compound (SMILES): Cc1c(-c2ccc(F)cc2)nnc(N2CCN(c3cnc(C(=O)O)cn3)[C@H](C)C2)c1C
InChIKey: LIOVPPURPDQFMB-CYBMUJFWSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 422.46, ALogP 3.1, HBA 7, HBD 1, PSA 95.34, QED 0.69
Compound (SMILES): CN(C)c1cccc2c(S(=O)(=O)N3CCC[C@H]3C(=O)N[C@@H](Cc3ccc(NC(=O)CCCCNC(=N)N)cc3)C(=O)O)cccc12
InChIKey: NHTSPCQURYFKRF-BDYUSTAISA-N
Target: Integrin alpha-V/beta-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Inhibition of integrin alphaVbeta1 (unknown origin)-mediated CHO cell adhesion to fibronectin preincubated for 15 to 30 mins followed by 60 min incubation for adhesion by crystal violet staining based cell adhesion assay
Measurement: IC50 = 79 nM (pChEMBL 7.099999904632568)
Descriptors: MW 651.79, ALogP 2.46, HBA 7, HBD 6, PSA 198.02, QED 0.09
Compound (SMILES): CC(C)(C)OC(=O)N1CCC2(CC1)CC2CNC(=O)NCc1cccnc1
InChIKey: PIVAZIMYJGDVBC-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Inhibition of NAMPT in human A2780 cells assessed as decrease in cell viability after 72 hrs by SRB assay
Measurement: IC50 > 2000 nM (pChEMBL 5.698999881744385)
Descriptors: MW 374.49, ALogP 2.92, HBA 4, HBD 2, PSA 83.56, QED 0.85
Compound (SMILES): CC(C)c1cc(CNc2nc(Nc3cc(C4CC4)[nH]n3)cc(N3CCN(C=O)CC3)n2)on1
InChIKey: LLYCEAVVAGEZLB-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 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 451.54, ALogP 2.82, HBA 9, HBD 3, PSA 128.1, QED 0.42
Compound (SMILES): CN1CCN(c2ccc(C(=O)Nc3n[nH]c4cc(OCc5cccc(Oc6ccccc6)c5)ccc34)cc2)CC1
InChIKey: ZEMPBQAQWWJUJQ-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Biochemical Assay: The assay has been performed using KIT cytoplasmic domain product and purified in house as GST fused protein. The KIT protein (4.5 microM) was pre activated with 300 microM ATP for 1 hour at 28*C in order to obtain a linear kinetic. Kinase buffer was composed of 50 mM HEPES pH 7.9 containing 5 mM MgCl2, 1 mM MnCl2, 10 mM DTT, 3 microM Na3VO4, and 0.2 mg/mL BSA. The KIT kinase assay was run with a final pre activated enzyme concentration of 4 nM, in the presence of 4.4 microM ATP (residual ATP from KIT pre activation step is negligible), 3.9 nM 33P-γ-ATP and 2.5 microM of substrate BioDB n*138 (Aminoacidic sequence: KWEEINGNNYVYIDPTQLPYDHKWEFPRNR- SEQ ID NO: 2).
Measurement: IC50 = 2619 nM (pChEMBL 5.579999923706055)
Descriptors: MW 533.63, ALogP 5.94, HBA 6, HBD 2, PSA 82.72, QED 0.26
Compound (SMILES): CC(C(N)=O)c1ccccc1CCc1nc(Nc2cn[nH]c2)ncc1Cl
InChIKey: RKVTUBMTXZINFU-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P35916
Assay: VEGFR3 Phospho ELISA Assay: Adult human dermal lymphatic microvascular endothelial cells (HMVEC-dLyAD) (Cat# CC-2810, Lonza) were seeded into clear-bottom, TC treated 12 well plates (Cat #665180, Greiner Bio-One) in EGM-2MV (Cat# CC-3202, Lonza) at 180,000 cells/well (volume 1 mL), and the plates incubated at 37° C. and 5% CO2 for 6 hours. The media was replaced with EBM-2 (Cat # CC-3156, Lonza)+0.1% BSA (Cat# A8412, Sigma) and cells incubated for a further period (overnight at 37° C. and 5% CO2).96 well Maxisorp immuno plates (Cat #439454, Nunc) were coated with 100 uL of Total VEGFR3 capture antibody (Part #841888, Human Total VEGFR3/FLT4 ELISA Kit, Cat # DYC3491, R&D Systems), or Phospho VEGFR3 Capture antibody (Part #841885, Human Phospho VEGFR3/FLT4 ELISA Kit, Cat# DYC2724, R&D Systems). The plates were covered and incubated at room temperature overnight .The coating antibody was flicked out and the plates washed three times with Wash Buffer (Phosphate buffered saline (137 mM NaCl, 2.7 nM KCl, 8.1 nM Na2HPO4, 1.5 mL KH2PO4, pH 7.2-7.4), 0.05% Tween 20). 300 uL of blocking buffer (5% v/v Tween 20, 5% w/v sucrose in PBS) was then added to wells and plate incubated for 2 hours at room temperature.
Measurement: IC50 = 64 nM (pChEMBL 7.190000057220459)
Descriptors: MW 370.84, ALogP 2.97, HBA 5, HBD 3, PSA 109.58, QED 0.59
Compound (SMILES): COc1cc(C(=O)N2CC[C@H]2c2nc(-c3ccccc3OC(F)(F)F)no2)c(-n2nccn2)cc1F
InChIKey: FLEPWMSNJHLEST-HNNXBMFYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Intracellular Calcium Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/mL G418, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM. Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 μL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 μM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement. Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 μL/well, incubated for 120 min and finally 10 μL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist.
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 504.4, ALogP 3.95, HBA 9, HBD 0, PSA 108.4, QED 0.36
Compound (SMILES): COCc1nc2c(c(Nc3ccc(C(F)(F)F)cc3)n1)CCN(c1ncccc1C(F)(F)F)CC2
InChIKey: XSJOKQIPEFOWGV-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 = 91 nM (pChEMBL 7.039999961853027)
Descriptors: MW 497.44, ALogP 5.4, HBA 6, HBD 1, PSA 63.17, QED 0.48
Compound (SMILES): OCC1CCN(c2ncnc3[nH]cc(-c4ccccc4)c23)CC1
InChIKey: CLTLWPSRLYCESA-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Lantha Screen Technology Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat # PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: 3 uL of a working solution of substrate (233 nM LRRKtide, 117 uM ATP) prepared in assay buffer (50 mM HEEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to a low volume Greiner 384-well plate. The compound dose response was prepared by diluting compound to a top concentration of 3.16 mM in 100% DMSO and serial diluted by half-log in DMSO 11 times. Aliquots (3.5 uL) of the 100% DMSO dose response were mixed with 46.5 uL water.
Measurement: IC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 308.39, ALogP 2.83, HBA 4, HBD 2, PSA 65.04, QED 0.78
Compound (SMILES): Cc1cn2ccc(N3CCC(OC(=O)N4CCN(C(C)C)CC4)CC3)cc2n1
InChIKey: XAQBWJISZRMNHF-UHFFFAOYSA-N
Target: Histamine H3 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5N1
Assay: Radioligand Binding Assay: The affinity of compounds of the invention to the H3 receptor can be assessed by measuring displacement of binding of the radioligand [3H]-N-Î±-Methylhistamine (PerkinElmer, #NET1027250UC) to membranes containing human H3 receptors (PerkinElmer, #ES-392-M400UA; GenBank: NM-007232.2; Hill S J et al, International Union of Pharmacology XIII. Classification of histamine receptors, Pharmacol Rev, 49(3), 253-278, 1997).Protocol: 1. Preparation of binding assay buffer (500 ml): 25 ml 1 M Tris-HCl pH 7.5 (Invitrogen, #15567-027), 2.5 ml 1 M MgCl2 (Sigma, #M1028-100ML), 472.5 ml ddH2O. 2. Compound serial dilution: Dilution was performed by BioTek Precision on compound dilution plate. Compound concentrations start at 5 or 10 uM, 10 point dose titrations with 3- or 5-fold serial dilutions. 3. Preparation of 2x membrane solution (25 ml): 1.25 ml human Histamine H3 receptor stock, 23.75 ml assay buffer. 4. Preparation of 2x solution of [3H]-N-Î±-methylhistamine (25 ml).
Measurement: Ki = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 385.51, ALogP 2.77, HBA 6, HBD 0, PSA 53.32, QED 0.81
Compound (SMILES): Cc1nc(C(NC(=O)c2ccc(N3CC(F)(F)C3)c(OCC3CC3)n2)C2CC2)no1
InChIKey: DTUQXJLSDVPMLV-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 419.43, ALogP 2.9, HBA 7, HBD 1, PSA 93.38, QED 0.7
Compound (SMILES): O=C1c2ccccc2C(O)(c2ccc3[nH]c(Nc4nccs4)nc3c2)N1c1cccc(Cl)c1F
InChIKey: ZTXVRLBTVUWHDH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 491.94, ALogP 5.41, HBA 6, HBD 3, PSA 94.14, QED 0.31
Compound (SMILES): CC(C)c1ccc2c(c1)C(SC(C)(C)C)C(CC(C)(C)C(=O)O)C2Cc1ccc(Cl)cc1
InChIKey: SYLBCYQWXLWMGW-UHFFFAOYSA-N
Target: DNA polymerase kappa (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBT6
Assay: Inhibition of human DNA polymerase kappa (19 to 526 residues) preincubated for 15 mins followed by replicating non-damaged DNA substrate addition measured after 30 mins in presence of dCTP and dGTP by radioactive gel-based primer extension assay
Measurement: IC50 = 13000 nM (pChEMBL 4.889999866485596)
Descriptors: MW 473.12, ALogP 8.49, HBA 2, HBD 1, PSA 37.3, QED 0.44
Compound (SMILES): COc1cc2c(Nc3ccc(Br)c(Cl)c3F)ncnc2cc1O[C@@H]1C[C@H]2OC[C@H](OC)[C@H]2O1
InChIKey: HDJUYMWWZOHAHK-WKRCXCSHSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: Inhibition of recombinant human N-terminal His6-tagged EphA2 (N598 to R890 residues) expressed in baculovirus using poly-EY as substrate after 180 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 540.77, ALogP 4.84, HBA 8, HBD 1, PSA 83.96, QED 0.44
Compound (SMILES): CCOc1cc(=O)n(C)cc1-c1cc(N(Cc2cn(C)cn2)S(=O)(=O)Cc2ccccc2)ccc1Oc1ccc(F)cc1F
InChIKey: ZWNHUJLAGSXGKS-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Assay method C: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution from one of the following: 0.047 mM to 0.78 nM or 5-fold serial dilution from Assay method A. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus# 6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe. The final concentration of 1X assay mixture for assay methods A, B and C contains 2% DMSO, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively) and compound concentration in the range of: 49.02 μM-15.63 nM for method A, 9.19 μM 150 pM for method B, and 0.92 μM 15 pM for method C. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 92.8 nM (pChEMBL 7.03000020980835)
Descriptors: MW 620.68, ALogP 5.79, HBA 8, HBD 0, PSA 95.66, QED 0.19
Compound (SMILES): Cc1cc(OCCCC(=O)O)ccc1-c1cc(OC(F)(F)F)ccc1Cl
InChIKey: IXCGAOKQTBZAKH-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Mus musculus
UniProt: Q7TMA4
Assay: Agonist activity at mouse GPR120 expressed in CHOK1 cells measured after 60 mins by IP1-HTRF assay
Measurement: EC50 = 78 nM (pChEMBL 7.110000133514404)
Descriptors: MW 388.77, ALogP 5.46, HBA 3, HBD 1, PSA 55.76, QED 0.64
Compound (SMILES): CN(CCCn1c(=O)[nH]c2cc(CNC[C@H](O)c3ccc(O)c4[nH]c(=O)ccc34)ccc21)[C@H]1CC[C@H](OC(=O)C(O)(c2cccs2)c2cccs2)CC1
InChIKey: LURMNNWUAQQCSQ-ZDQHWEPJSA-N
Target: Muscarinic acetylcholine receptor M3 (SINGLE PROTEIN), Homo sapiens
UniProt: P20309
Assay: Muscarinic Receptor Binding Assay: The study of binding to human muscarinic M1, M2, M3, M4 and M5 receptors was performed using commercial membranes (Perkin Elmer) prepared from CHO-K1 cells. Radioligand binding experiments were conducted in 96 polypropylene well plates in a total volume of 200 ul. All reagents were dissolved in assay binding buffer (PBS with calcium and magnesium, SIGMA), except compounds that were dissolved in DMSO 100%. Non-specific binding (NSB) was measured in the presence of 1 uM atropine. [3H]-NMS was used as the radioligand at a concentration of 1 nM for M2, M3 and M5 and 0.3 nM for M1 and M4. [3H]-NMS and antagonists were incubated with membranes that express human muscarinic receptors M1, M2, M3, M4 and M5 at concentrations of 8.1, 10, 4.9, 4.5 and 4.9 ug/well, respectively. After an incubation period of two hours with gentle shaking, 150 ul of the reaction mix were transferred to 96 GF/C filter plates (Millipore), previously treated with wash buffer.
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 757.94, ALogP 4.94, HBA 12, HBD 6, PSA 172.91, QED 0.08
Compound (SMILES): CCOc1ccccc1C(=O)NCCCCCC(=O)Nc1ccccc1N
InChIKey: PMCFEYQREKJMPI-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 338 nM (pChEMBL 6.46999979019165)
Descriptors: MW 369.46, ALogP 3.6, HBA 4, HBD 3, PSA 93.45, QED 0.44
Compound (SMILES): COc1cc(C(=O)NCc2cccc(C(=O)NCC(C)(C)O)c2)ccc1-c1cn[nH]c1
InChIKey: WHVMMFVGEVMVIT-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 0.46 nM (pChEMBL 9.34000015258789)
Descriptors: MW 422.49, ALogP 2.52, HBA 5, HBD 4, PSA 116.34, QED 0.45
Compound (SMILES): Clc1cccc2c1N1CCCNCCC1CC2
InChIKey: UAPJPSLLIFFBKM-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2B (SINGLE PROTEIN), Homo sapiens
UniProt: P41595
Assay: Agonist activity at 5-HT2B receptor (unknown origin) expressed in HEK293 cells assessed as [3H]inositol phosphate accumulation after 2 hrs by scintillation counting
Measurement: EC50 = 100 nM (pChEMBL 7)
Descriptors: MW 250.77, ALogP 2.84, HBA 2, HBD 1, PSA 15.27, QED 0.76
Compound (SMILES): CN1CCC(Nc2ccc(S(=O)(=O)NC(=O)c3ccc(N4CCN(C/C5=C(\c6ccc(Cl)cc6)CCCCCC5)CC4)cc3Oc3cccc4[nH]ccc34)cc2[N+](=O)[O-])CC1
InChIKey: QTIWKELYIHAPRB-GUPKEBGXSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
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 > 660 nM (pChEMBL 6.179999828338623)
Descriptors: MW 866.48, ALogP 9.08, HBA 10, HBD 3, PSA 153.15, QED 0.08
Compound (SMILES): C=C1C[C@@](C)(c2cc(NC(=O)c3ccc(C#N)cn3)ccc2F)N=C(N)S1
InChIKey: QQOKHWYWSYNBSI-IBGZPJMESA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Inhibition of BACE1 in human SH-SY5Y cells expressing wild type betaAPP assessed as reduction in amyloidbeta (1 to 40 residues) level incubated for 24 hrs by HTRF assay
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 381.44, ALogP 3.53, HBA 6, HBD 2, PSA 104.16, QED 0.85
Compound (SMILES): CC(=C(c1ccc(N)cc1)c1ccc(NCC(N)=O)cc1)c1ccc([N+](=O)[O-])cc1
InChIKey: VJRVGNMDZPKEME-UHFFFAOYSA-N
Target: Aromatase (SINGLE PROTEIN), Homo sapiens
UniProt: P11511
Assay: Inhibition of recombinant human microsomal CYP19 using MFC as substrate measured after 30 mins by fluorometric analysis
Measurement: IC50 = 645.3 nM (pChEMBL 6.190000057220459)
Descriptors: MW 402.45, ALogP 4.05, HBA 5, HBD 3, PSA 124.28, QED 0.24
Compound (SMILES): COc1cc(-c2cn(C)nc2C)ccc1Nc1ncc2ccnc(NCC(C)(C)C)c2n1
InChIKey: LEVJITMIUUMAFY-UHFFFAOYSA-N
Target: CDK2/Cyclin A2 (PROTEIN COMPLEX), Homo sapiens
UniProt: P20248
Assay: Inhibition of full length human N-terminal His-tagged CDK2/cyclinA expressed in baculovirus expression system using 5FAM- peptide18 as substrate after 60 mins by caliper assay
Measurement: IC50 = 2800 nM (pChEMBL 5.550000190734863)
Descriptors: MW 431.54, ALogP 4.94, HBA 8, HBD 2, PSA 89.78, QED 0.45
Compound (SMILES): CC(c1ccccc1)c1ccc(O)cc1O
InChIKey: PQSXNIMHIHYFEE-UHFFFAOYSA-N
Target: Tyrosinase (SINGLE PROTEIN), Homo sapiens
UniProt: P14679
Assay: Inhibition of human His-tagged tyrosinase expressed in HEK 293 cells using L-DOPA as substrate by Lineweaver-Burk plot analysis
Measurement: Ki = 24000 nM (pChEMBL 4.619999885559082)
Descriptors: MW 214.26, ALogP 3.25, HBA 2, HBD 2, PSA 40.46, QED 0.81
Compound (SMILES): CCOC(=O)O[C@@H](C)OC(=O)c1c(-c2ccc(C(=O)N(c3ncccc3Cl)[C@@H]3CCCNC3)cc2)cnn1C
InChIKey: DUHCQXCKCLIECZ-FXAWDEMLSA-N
Target: Proprotein convertase subtilisin/kexin type 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NBP7
Assay: Cell Free Assay: In order to eliminate the permeability barrier inherent to the WT7 and SCHH cell-based assays a cell-free system was also established to access compound activity. A sequence containing the full length human PCSK9 (NCBI reference identifier, NM-174936.3, where coding sequence start annotated at position 363) along with 84 additional 3â¿² nucleotides, comprising a V5 tag and polylinkinker followed by an in frame modified firefly luciferase reporter (corresponding to nucleotide positions 283-1929 of pGL3, Gen Bank reference identifier JN542721.1) was cloned into the pT7CFE1 expression vector (ThermoScientific). The construct was then in-vitro transcribed using the MEGAscript T7 Kit (Life Technologies) and RNA subsequently purified incorporating the MEGAclear Kit (Life Technologies) according to manufacturer's protocols. HeLa cell lysates were prepared following the protocols described by Mikami (reference is Cell-Free Protein Synthesis Systems with Extracts from Cultured Human Cells, S. Mikami, T. Kobayashi and H. Imataka; from Methods in Molecular Biology, vol. 607, pages 43-52, Y. Endo et al. (eds.), Humana Press, 2010) with the following modifications.
Measurement: IC50 = 3100 nM (pChEMBL 5.510000228881836)
Descriptors: MW 556.02, ALogP 4.21, HBA 10, HBD 1, PSA 124.88, QED 0.32
Compound (SMILES): Cc1cccc2cc(Cn3nc(-c4cn[nH]c4)c4c(N)ncnc43)n(-c3cccc(F)c3)c(=O)c12
InChIKey: ZCGSFTXYADUZTH-UHFFFAOYSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 466.48, ALogP 3.6, HBA 8, HBD 2, PSA 120.3, QED 0.41
Compound (SMILES): CCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)CNC(=O)[C@H](CC(=O)O)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc1ccccc1)NC(C)=O)C(C)C)C(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O
InChIKey: DRBBMCJNAIAQLF-URURHIFTSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Inhibition of C-terminus 10x-His-taged human TFPI-1 (29 to 282 residues) assessed as inhibition of factor 10a using S-2222 substrate incubated for 115 mins by micro-plate reader based assay
Measurement: EC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 2358.73
Compound (SMILES): C[C@@]1(c2cc(Nc3nccc4cc(C#N)cnc34)cc(F)c2F)C[C@@H](C(F)(F)F)OC(N)=N1
InChIKey: SWFXRUVBVUKMPN-YWZLYKJASA-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 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 462.38, ALogP 4.4, HBA 7, HBD 2, PSA 109.21, QED 0.56
Compound (SMILES): O=C(NO)C1CCN(CCOc2ccc3cc2COC/C=C/COCc2cccc(c2)-c2ccnc(n2)N3)CC1
InChIKey: HFQVERAALUPSOG-OWOJBTEDSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Inhibition of full length human recombinant N-terminal GST-tagged HDAC6 (1 to 1215 residues) expressed in sf9 cells preincubated with enzyme followed by fluorogenic Arg-His-Lys-Lys(Ac)-AMC substrate addition measured after 2 hrs by fluorescence assay
Measurement: IC50 = 899 nM (pChEMBL 6.050000190734863)
Descriptors: MW 545.64, ALogP 4.09, HBA 9, HBD 3, PSA 118.07, QED 0.25
Compound (SMILES): Cc1cc(-c2cn(CCN3CCC3)c(C3CCN(c4ncnc(N)c4C#N)CC3)n2)ccc1F
InChIKey: GIMRLCRHTAIQJM-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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 460.56, ALogP 3.33, HBA 8, HBD 1, PSA 99.89, QED 0.6
Compound (SMILES): CNCc1ccc(Cl)c(-c2ccccc2OC)c1
InChIKey: NSASZAUFRUOUIN-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P34969
Assay: Agonist activity at human 5-HT7R expressed in HEK293 cells assessed as induction of cAMP levels treated for 10 mins measured after 30 mins by D2-dye based fluorescence assay
Measurement: EC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 261.75, ALogP 3.74, HBA 2, HBD 1, PSA 21.26, QED 0.91
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CCC(NCc2ccncc2)CC1
InChIKey: ABAWAKYNDLSEJP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 1004 nM (pChEMBL 6)
Descriptors: MW 448.51, ALogP 3, HBA 8, HBD 3, PSA 134.54, QED 0.45
Compound (SMILES): O=C(N[C@H]1CCC[C@@H]1Nc1ccc(C(F)(F)F)cn1)c1ccccc1Cl
InChIKey: GWLZLMIQIQBKLX-GJZGRUSLSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Orexin antagonist activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by Fluorescent Imaging Plate Reader (FLIPR®) technology available from Molecular Devices, LLC, U.S.A. Orexin mediated increases in intracellular Ca2+ concentration were readily detected upon activation with orexin-A. Twenty-four hours prior to the assay, RBL-2H3 cells stably expressing either human orexin receptor 1 or human orexin receptor 2 were seeded in cell culture medium in black, clear-bottom 384-well plates (commercially available from Corning Inc., U.S.A.) and grown overnight at 37 °C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (commercially sold by Molecular Devices, LLC, U.S.A.) for 1 hour at 37 °C., 5% CO2.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 383.8, ALogP 4.52, HBA 3, HBD 2, PSA 54.02, QED 0.82
Compound (SMILES): CNCCN1CCC(NC(=O)c2ccccc2)CC1
InChIKey: GHEBNMWUAGLPPM-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96LA8
Assay: Inhibition of human full length PRMT6 (1 to 375 residues) expressed in baculovirus expression system assessed as inhibition of methylation activity preincubated with protein followed by addition of biotin labeled histone H4 (1 to 24) peptide as substrate and [3H]-SAM measured after 3 hrs by scintillation proximity assay
Measurement: IC50 = 129 nM (pChEMBL 6.889999866485596)
Descriptors: MW 261.37, ALogP 1.1, HBA 3, HBD 2, PSA 44.37, QED 0.83
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)Nc3cc(F)ccc3F)CC2)C1
InChIKey: CIVCERVVQBMFGU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 517.54, ALogP 3.72, HBA 7, HBD 2, PSA 118.76, QED 0.42
Compound (SMILES): N#Cc1cc(-c2ccnc(Nc3cccc(=O)[nH]3)n2)cnc1N1CC[C@H](F)C1
InChIKey: VETBWYSGFIOLHB-AWEZNQCLSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 377.38, ALogP 2.39, HBA 7, HBD 2, PSA 110.59, QED 0.72