lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(=O)N1CCc2c(c(Nc3cccc(C)c3)nn2Cc2ccncc2)C1
InChIKey: ZUDONVCZBCQPST-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaLisa Binding Assay: His/Flag epitope tagged BRD4 BD142-168 was cloned, expressed, and purified to. BRD4 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-1/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD4(BD1) (30 nM final) was combined with peptide (200 nM final) in 40 mM HEPES (pH 7.0), 40 mM NaCl, 1 mM DTT, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 1.2% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature Alpha streptavidin donor beads and AlphaLisa anti-Flag acceptor beads were added to a final concentration of 10 ug/mL each. After three hours equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 5190 nM (pChEMBL 5.289999961853027)
Descriptors: MW 361.45, ALogP 3.28, HBA 5, HBD 1, PSA 63.05, QED 0.77
Compound (SMILES): C[C@@H](Nc1ncnc2[nH]c(-c3ccc(CN4CCN(CCCCCCC(=O)NO)CC4)cc3)cc12)c1ccccc1
InChIKey: NDRDPOLTZPQUOD-XMMPIXPASA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of N-terminal GST-His6-thrombin cleavage site-fused human VEGFR2 (Asp805 to Val1356 residues) expressed in baculovirus expression system using biotinylated gastrin precursor at Tyr87 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA method
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 555.73, ALogP 5.37, HBA 7, HBD 4, PSA 109.41, QED 0.1
Compound (SMILES): CCN(c1cc(Cl)cc(C(=O)NCc2c(C)cc(C)[nH]c2=O)c1C)C1CCC(CO)CC1
InChIKey: DEGTXGLOIUINEP-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 460.02, ALogP 4.26, HBA 4, HBD 3, PSA 85.43, QED 0.58
Compound (SMILES): Cc1c(Cc2c(CNCCS(C)(=O)=O)nc3c(N)cc(N4CCOCC4)nn23)cccc1C(F)(F)F
InChIKey: GWFUHMDGIDDKAR-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Binding affinity to human PI3Kdelta (108 to 1044 residues) expressed in mammalian expression system by KINOMEscan assay
Measurement: Kd = 178 nM (pChEMBL 6.75)
Descriptors: MW 526.59, ALogP 2.2, HBA 9, HBD 2, PSA 114.85, QED 0.43
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3ccc(-c4ccccc4)cc3)CC2)C1
InChIKey: DUKGQZXOHHYYOE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In vitro JAK Kinase Assay: 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 = 12.5 nM (pChEMBL 7.900000095367432)
Descriptors: MW 542.65, ALogP 4.72, HBA 7, HBD 1, PSA 106.73, QED 0.33
Compound (SMILES): FC(F)(F)C1(NC[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)CC1
InChIKey: KXXAZUVPEFTPQM-HAQNSBGRSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 = 390 nM (pChEMBL 6.409999847412109)
Descriptors: MW 378.4, ALogP 3.56, HBA 5, HBD 2, PSA 70.9, QED 0.73
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(-c3ccc4cc[nH]c4c3)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: VGAJKHFCGZSKTP-UHFFFAOYSA-N
Target: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110alpha/untagged recombinant full length human 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 > 10000 nM (pChEMBL 5)
Descriptors: MW 511.59, ALogP 5.53, HBA 7, HBD 2, PSA 107.41, QED 0.32
Compound (SMILES): Cc1cc(Cl)c2c(C(=O)OCC34CCN(CC3)CC4)c[nH]c2c1
InChIKey: DCZGNMUEAXUIHE-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition Assay: hERG study was conducted with an automated patch clamp machine, Qpatch-48HT (Sophion Biosciences, Denmark). Cultured CHO cells stably expressing hERG channels (provided by Sophion Biosciences, Denmark) were harvested from culture flasks of 70-90% cell confluence rate and prepared as cell suspension with a cell density of 3-8&timse;10^6 cells/mL in serum-free media (CHO-S-SFM II, cat#12052 Invitrogen; 25 mM HEPES). Cells in such condition were placed into a Qpatch cell stir chamber and used within 4 hours. For each run, cells were first spun down by the built-in Qpatch centrifuge and re-suspended in an extracellular solution (in mM, 2 CaCl2, 1 MgCl2, 4 KCl, 145 NaCl, 10 Glucose, 10 HEPES, pH 7.4, osmolarity ~305 mOsm). Qplate-48 that holds one cell in each of its 48 channels for the later voltage-clamped assay were primed with the extracellular solution and intracellular solution (in mM, 5.4 CaCl2, 1.75 MgCl2, 120 KCl, 10 HEPES, 5 EGTA, 4 NaATP, pH 7.25, Osmolarity ~280-295 mOsm). Cells were dispatched by Qpatch robotic dispensing guns into each Qplate channel and went through the process of giga-ohm sealing and whole cell configuration. Whole-cell recordings were performed in voltage-clamp mode at a holding potential of −80 mV. The hERG current was activated by depolarizing at +20 mV for 5 sec, after which the current was taken back to −50 mV for 5 sec to remove the inactivation and observe the deactivating tail current. The maximum amount of tail current size was used to determine the hERG current amplitude. The above voltage protocol was applied to the cells every 15 sec throughout the whole procedure. External solution containing 0.1% DMSO (vehicle) was applied to the cells to establish the baseline. Compound solution was added, and the cells were kept in the test solution until the compound's effect reached a steady state or for a maximum of 4 min. For dose response assays (0.1, 0.3, 1, 3, 10 and 30 μM), the compound was applied to the cells accumulatively from low to high concentrations. Washout with extracellular solution was performed after compound testing. Positive control cisapride 0.1 μM was used on each cell after compound testing to ensure the normal response and the good quality of the cell.
Measurement: IC50 = 2430 nM (pChEMBL 5.610000133514404)
Descriptors: MW 332.83, ALogP 3.77, HBA 3, HBD 1, PSA 45.33, QED 0.87
Compound (SMILES): O=C(c1cccc(Cl)c1-n1nccn1)N1CCOC[C@H]1Cc1cccc(-n2nccn2)c1
InChIKey: IGNKAYVYFKMOBL-GOSISDBHSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: CYP3A4 Inhibition Assay: The CYP3A4 inhibition assay was performed using human liver microsomes and testosterone 6'-hydroxylation as a P450 isoform-specific marker. In a total volume of 150 uL, 14C-testosterone at a final concentration of 40 uM in a 100 mM phosphate buffer (pH 7.4) was incubated in a 96-well plate with 0.3 mg/mL of human liver microsomes in an Eppendorf thermomixer at 37° C. and 400 rpm. A 1.0 uL-aliquot of the 150-fold concentrated compound stock solution, prepared in DMSO, was added to yield final inhibitor concentrations of 0, 0.1, 0.5, 1.0, 5.0, 10, 25, and 50 uM. The reaction was initiated by addition of 15 uL of the NADPH-regenerating system containing the glucose-6-phosphate dehydrogenase and terminated after 7 min with a 75 uL-aliquot of methanol. After centrifugation at 465 g and 4° C. for 20 min, a 50 uL-aliquot of the supernatant was submitted to HPLC according to the method described below.
Measurement: IC50 = 22000 nM (pChEMBL 4.659999847412109)
Descriptors: MW 449.9, ALogP 2.59, HBA 8, HBD 0, PSA 90.96, QED 0.46
Compound (SMILES): O=C(O)CCC1=CC[C@@H]2[C@@H](/C=C/[C@@H](O)COc3ccccc3)[C@H](O)C[C@@H]2OC1
InChIKey: HTUHVPVHRIFBLU-DCFAJTMGSA-N
Target: Prostacyclin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43119
Assay: Agonist activity at human IP receptor expressed in CHO cells assessed as increase in intracellular cAMP level by HTRF method
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 388.46, ALogP 2.56, HBA 5, HBD 3, PSA 96.22, QED 0.59
Compound (SMILES): CC[C@H]1C(=O)C[C@@H]2CCN(S(=O)(=O)c3ccc(C)cc3)[C@@H]3CCC[C@H]1[C@@H]23
InChIKey: PZMUSBZBIIMWES-PPVISPMTSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Displacement of [3H]DAMGO from human MOR expressed in HEK cells after 90 mins by microbeta scintillation counting method
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 375.53, ALogP 3.79, HBA 3, HBD 0, PSA 54.45, QED 0.81
Compound (SMILES): O=C(CNC[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1)NCC(F)(F)F
InChIKey: XPNMGPMMJXKVHV-HAQNSBGRSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 = 310 nM (pChEMBL 6.510000228881836)
Descriptors: MW 409.42, ALogP 2.15, HBA 6, HBD 3, PSA 100, QED 0.58
Compound (SMILES): COc1cc(NC(=O)OCCN(C)[C@H]2CC[C@H](OC(=O)C(O)(c3cccs3)c3cccs3)CC2)c(Cl)cc1CNC[C@H](O)c1ccc(O)c2[nH]c(=O)ccc12
InChIKey: SNXJYSXRLKUBSZ-WBXFWKJNSA-N
Target: Beta-2 adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07550
Assay: Agonist activity at human beta2 adrenoceptor
Measurement: EC50 = 0.2512 nM (pChEMBL 9.600000381469727)
Descriptors: MW 811.38, ALogP 6.11, HBA 13, HBD 6, PSA 182.68, QED 0.07
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCc1noc(C2CCCN(C)C2)n1
InChIKey: WUCSNTVXKXMTBP-UHFFFAOYSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 = 525 nM (pChEMBL 6.28000020980835)
Descriptors: MW 515.4, ALogP 5.46, HBA 9, HBD 1, PSA 98.43, QED 0.34
Compound (SMILES): Cc1cc(F)c(C(=O)Nc2cccc(-c3nncn3C(C)C)n2)cc1-n1cnc(C2CC2)c1
InChIKey: YIDDLAAKOYYGJG-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: Inhibition of ASK1 (unknown origin)
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 445.5, ALogP 4.68, HBA 7, HBD 1, PSA 90.52, QED 0.47
Compound (SMILES): N#Cc1cnc2[nH]cc(C(=O)c3c(F)ccc(NS(=O)(=O)c4ccccc4)c3F)c2c1
InChIKey: LZPYPWMVRFVWBG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf V600E mutant (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 438.42, ALogP 3.74, HBA 5, HBD 2, PSA 115.71, QED 0.46
Compound (SMILES): COc1cccc(-c2ccc3c(c2)[C@@]2(N=C(C)C(N)=N2)[C@]2(CC[C@H](OC)CC2)C3)c1F
InChIKey: MUPBWXCOUHZWEH-WDNCENIBSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Diluted TR-FRET Assay: TR-FRET AssayThe beta-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human beta-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 ul) was added to the plate then 1 ul of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes.
Measurement: IC50 = 3.3 nM (pChEMBL 8.479999542236328)
Descriptors: MW 421.52, ALogP 4.62, HBA 5, HBD 1, PSA 69.2, QED 0.79
Compound (SMILES): Cc1cc(C)nc(-n2nnc3c2CCN(C(=O)c2cccc(C(F)(F)F)c2Cl)C3C)c1
InChIKey: HBOLBGHPFJCVDU-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 20230.2 nM (pChEMBL 4.690000057220459)
Descriptors: MW 449.86, ALogP 4.71, HBA 5, HBD 0, PSA 63.91, QED 0.57
Compound (SMILES): Fc1cccc(-c2cc3nc4c(c(N5CCNCC5)n3n2)CCCC4)c1
InChIKey: AFYBBCQVYAHRRH-UHFFFAOYSA-N
Target: Beta-glucuronidase (SINGLE PROTEIN), Homo sapiens
UniProt: P08236
Assay: Cell-Based Assay: PheG Assay: Two ß-glucuronidase activity assays were employed to examine the potency of inhibitors both in vitro and in cell-based studies. An absorbance assay based on the conversion of p-nitrophenyl glucuronide (PNPG) to p-nitrophenol (PNP) (Szasz (1967) Clin Chem 13:752-759), which absorbs at 410 nm, was employed as the primary in vitro assay. A secondary assay involving the conversion of phenolphthalein glucuronide (PheG) to phenolphthalein (Phe), which absorbs at 540 nm, was also employed. Id. Both assays were validated in vitro and in living cells, and the wavelengths monitored did not overlap with absorbance characteristics of putative inhibitors.
Measurement: IC50 = 1266.4 nM (pChEMBL 5.900000095367432)
Descriptors: MW 351.43, ALogP 2.82, HBA 5, HBD 1, PSA 45.46, QED 0.77
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCC1CN(Cc2ccccn2)CCO1
InChIKey: KOHLHBQFFZUSET-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 526.42, ALogP 5.36, HBA 8, HBD 1, PSA 81.63, QED 0.33
Compound (SMILES): COc1ccc(/C=C/C(=O)NCCC2CCN(Cc3ccccc3)CC2)c(OC)c1
InChIKey: AHWHBOQIIOPNCM-PKNBQFBNSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: Inhibition of human recombinant MAOA using p-tyramine as substrate incubated for 15 mins by fluorimetric method
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 408.54, ALogP 4.14, HBA 4, HBD 1, PSA 50.8, QED 0.63
Compound (SMILES): CNC(=O)N=S(C)(=O)Cc1cccc(Nc2ncc(F)c(-c3ccc(F)cc3OC)n2)c1
InChIKey: SOSTXYOXUQYPHG-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: CDK9/CycT1 : Recombinant full-length His-tagged human CDK9 and CycT1, expressed in insect cells and purified by Ni-NTA affinity chromatography, were purchase from Invitrogen (Cat. No PV4131). As substrate for the kinase reaction biotinylated peptide biotin-Ttds-YISPLKSPYKISEG (C-terminus in amid form) was used which can be purchased e.g. form the company JERINI peptide technologies (Berlin, Germany) For the assay 50 nl of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 ul of a solution of CDK9/CycT1 in aqueous assay buffer [50 mM Tris/HCl pH 8.0, 10 mM MgCl2, 1.0 mM dithiothreitol, 0.1 mM sodium ortho-vanadate, 0.01% (v/v) Nonidet-P40 (Sigma)] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction.
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 461.49, ALogP 4.11, HBA 6, HBD 2, PSA 105.57, QED 0.57
Compound (SMILES): CN(C)[C@H]1c2cc3ccn(S(=O)(=O)c4ccccc4)c3cc2[C@H]1CCO
InChIKey: OCKYRBSJWIYEBM-OXQOHEQNSA-N
Target: 5-hydroxytryptamine receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P50406
Assay: Displacement of [3H]-LSD from human 5-HT6R expressed in CHO cell membranes by radioligand binding assay
Measurement: Ki = 39.81 nM (pChEMBL 7.400000095367432)
Descriptors: MW 370.47, ALogP 2.96, HBA 5, HBD 1, PSA 62.54, QED 0.75
Compound (SMILES): CN1CCC(Nc2ccc(S(=O)(=O)NC(=O)c3ccc(N4CCN(CC5=C(c6ccc(Cl)cc6)CC(C)(C)CC5)CC4)cc3Oc3ccc4[nH]ccc4c3)cc2[N+](=O)[O-])CC1
InChIKey: RRVGGZQEEIZHGA-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki = 0.013 nM (pChEMBL 10.890000343322754)
Descriptors: MW 866.48, ALogP 8.93, HBA 10, HBD 3, PSA 153.15, QED 0.08
Compound (SMILES): COc1cc2c(N[C@H]3COC[C@@H]3OC)ncnc2cc1O[C@H]1CO[C@H]2[C@@H]1OC[C@@H]2O
InChIKey: XRRHFQQSBXKXCI-FXELPECLSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 419.43, ALogP 0.37, HBA 10, HBD 2, PSA 113.42, QED 0.68
Compound (SMILES): COC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccccc1)NC(=O)CNC(=O)c1ccccc1
InChIKey: SSBGKJYMHDNYHV-QALZTRBASA-N
Target: Chromobox protein homolog 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HC52
Assay: Antagonist activity at recombinant human N-terminal His-tagged CBX8 chromodomain (8 to 61 residues) expressed in Escherichia coli Rosetta BL21(DE3)pLysS using biotin-labeled H3K9me3 measured after 30 mins by AlphaScreen assay
Measurement: IC50 > 60000 nM (pChEMBL 4.2220001220703125)
Descriptors: MW 739.87, ALogP -0.56, HBA 10, HBD 8, PSA 247.15, QED 0.06
Compound (SMILES): Cn1ncc(-c2cc3c(cn2)cnn3-c2cccc(N3CCCNCC3)n2)cc1=O
InChIKey: QXHXZOQHNCKQSX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 45.2 nM (pChEMBL 7.340000152587891)
Descriptors: MW 402.46, ALogP 1.38, HBA 9, HBD 1, PSA 93.76, QED 0.55
Compound (SMILES): CCn1nc(-c2cccc(-c3cccc(C(=O)NCCN4CCNCC4)c3)c2)cc(NC(=O)Nc2c(Cl)cncc2Cl)c1=O
InChIKey: FEAUWLMHIGKLBB-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Inhibition of human recombinant PDE4B1 assessed as reduction in [3H]cAMP hydrolysis to [3H]AMP incubated for 60 mins by PDE-SPA assay
Measurement: IC50 = 0.38 nM (pChEMBL 9.420000076293945)
Descriptors: MW 635.56, ALogP 4.58, HBA 8, HBD 4, PSA 133.28, QED 0.21
Compound (SMILES): O=C(Nc1cccc(Nc2ncc(Br)c(NCCc3cnc[nH]3)n2)c1)N1CCCC1
InChIKey: DMMILYKXNCVKOJ-UHFFFAOYSA-N
Target: MAP/microtubule affinity-regulating kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96L34
Assay: Displacement of tracer K5 from NLuc fused MARK4 (unknown origin) expressed in HEK293 cells by NanoBRET assay
Measurement: IC50 = 338 nM (pChEMBL 6.46999979019165)
Descriptors: MW 471.36, ALogP 3.99, HBA 6, HBD 4, PSA 110.86, QED 0.41
Compound (SMILES): COc1ccc(CCO[C@H]2CCCC[C@@H]2N2CC[C@@H](O)C2)cc1OC.Cl
InChIKey: JMHYCBFEEFHTMK-PWGAQZMISA-N
Target: Potassium voltage-gated channel subfamily A member 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P22460
Assay: Inhibition of human KV1.5 expressed in HEK cells by patch clamp method
Measurement: IC50 = 6000 nM (pChEMBL 5.21999979019165)
Descriptors: MW 349.47, ALogP 2.64, HBA 5, HBD 1, PSA 51.16, QED 0.82
Compound (SMILES): CN(C)CCn1cc(-c2ccc(F)c(C(F)(F)F)c2)nc1C1CCN(c2ncnc(N)c2C#N)CC1
InChIKey: WCBMSENIXPZAJT-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 502.52, ALogP 3.9, HBA 8, HBD 1, PSA 99.89, QED 0.51
Compound (SMILES): COc1cc(Nc2ccc(S(=O)(=O)NCC3CCCO3)c(C(F)(F)F)c2)cc(OC)c1
InChIKey: CPPNFVSJYCMUPR-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Binding Assay: The binding assays were performed by testing the ability of the new compounds to displace the radiolabeled synthetic non-selective cannabinoid agonist [3H]CP55940 (168 Ci/mmol; PerkinElmer) from the human CB1 (hCB1) or human CB2 (hCB2) receptors on membranes derived from stably transfected HEK-293 cells (PerkinElmer). Membranes were diluted in assay buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM MgCl2, 1 mg/ml BSA, pH=7.4). The amount of membrane was determined for each batch of membranes according to protein binding assay. The minimum amount of membrane that gave 50% specific binding was used for the binding assay. In most assays, binding was tested using 8 μg and 4 μg protein of hCB1 and hCB2 membranes, respectively. The tested compounds were dissolved in DMSO and diluted in assay buffer to a final concentration of 0.1% solvent. Total binding of [3H]CP55940 was evaluated with 1.5 nM to hCB1 and with 0.5 nM to hCB2, according to Kd affinity of [3H]CP55940 for the respective membranes. The ability of the tested compounds to displace [3H]CP55940 was evaluated first at a single concentration point of 100 nM for binding toward hCB2 or hCB1. In certain cases, the displacement was tested at compound concentrations ranging from 0.03 nM to 10 μM. Non-specific binding was measured by the addition of 1 μM of unlabelled CP55940 to the tubes. Binding assays were performed in triplicate in a total volume of 500 μl for 60 minutes at 30° C., in a shaking bath. Free and bound radioligands were separated by rapid filtration through GF/C filter plates (PerkinElmer) that had been presoaked with 0.1% Polyethylenimine (Sigma). Filters were shaken for 1 hour in 7 ml scintillation fluid (PerkinElmer) and radioactivity was determined by liquid scintillation counter (Wallac; PerkinElmer).
Measurement: Ki = 735 nM (pChEMBL 6.130000114440918)
Descriptors: MW 460.47, ALogP 3.92, HBA 6, HBD 2, PSA 85.89, QED 0.62
Compound (SMILES): CC1(O)CCC(NC(=O)c2cnn3ccc(N4CCC[C@@H]4c4cc(F)ccc4F)nc23)CC1
InChIKey: CYJPVTPYPTUQAC-HYSRAYMDSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Jak2 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit Jak2 using the general enzyme inhibition assay method, in which the assay mixture contained 500 μM ATP, 10 μM Omnia® Y7 peptide (Catalog # IVGN KNZ3071C, Invitrogen Corporation, Carlsbad, Calif.) and 4 nM Jak2 in a total volume of 20 μL. Human Jak2 kinase domain comprising amino acids 808-1132 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4210).
Measurement: IC50 = 1.18 nM (pChEMBL 8.930000305175781)
Descriptors: MW 455.51, ALogP 3.77, HBA 6, HBD 2, PSA 82.76, QED 0.63
Compound (SMILES): CC(C)Cc1sc2ccccc2c1C(=O)c1cccc2ccccc12
InChIKey: UPTNLTUDMBVVFL-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: Ki = 700 nM (pChEMBL 6.159999847412109)
Descriptors: MW 344.48, ALogP 6.48, HBA 2, HBD 0, PSA 17.07, QED 0.39
Compound (SMILES): CC(=S)NCCCC[C@@H]1NC(=O)[C@H](Cc2ccc3ccccc3c2)NC(=O)CCCCCCC(=O)NCC[C@@H](C(N)=O)NC1=O
InChIKey: KSQYPHZPLPZVKP-KCHLEUMXSA-N
Target: NAD-dependent protein deacetylase sirtuin-3, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NTG7
Assay: Inhibition of recombinant human His6-tagged SIRT3 using H2N-HK-[Nepsilon-acetyl-lysine]-LM-COOH as substrate incubated for 10 mins in presence of beta-NAD+ by HPLC based assay
Measurement: IC50 = 10800 nM (pChEMBL 4.96999979019165)
Descriptors: MW 638.84, ALogP 2.29, HBA 6, HBD 6, PSA 171.52, QED 0.19
Compound (SMILES): FC(F)Cn1cc(-c2cc3c(cn2)cnn3-c2cccc(N3CCCNCC3)n2)cn1
InChIKey: ZHIKDHHAKGWUBZ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.814 nM (pChEMBL 9.09000015258789)
Descriptors: MW 424.46, ALogP 2.74, HBA 8, HBD 1, PSA 76.69, QED 0.53
Compound (SMILES): COc1cc(OC)cc(N(CC#Cc2ccc(C#N)cc2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: HVEVXOZVOPQEHH-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 500.56, ALogP 5.11, HBA 8, HBD 0, PSA 89.09, QED 0.3
Compound (SMILES): C[C@@H]1CN(Cc2ccccc2)c2nc(C(F)(F)F)ccc2[C@H](C)N1C(=O)NCc1ccccc1
InChIKey: PZZIZFJADLSSCF-MOPGFXCFSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1I (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P0X4
Assay: Channel blocking activity at recombinant human CaV 3.3 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 = 281 nM (pChEMBL 6.550000190734863)
Descriptors: MW 468.52, ALogP 5.78, HBA 3, HBD 1, PSA 48.47, QED 0.53
Compound (SMILES): FC(F)(F)c1ccc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: WFPPWRAUXJMGTG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 = 78 nM (pChEMBL 7.110000133514404)
Descriptors: MW 400.41, ALogP 4, HBA 5, HBD 1, PSA 62.11, QED 0.56
Compound (SMILES): CC(=O)N1CCC(Nc2ncc(C(=O)NO)cn2)(C(C)C)CC1
InChIKey: HIMUFBIQHKSCQA-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC Enzyme Assay: The compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 uM TCEP) to 6 fold their final concentration. The HDAC enzymes (purchased from BPS Biosciences) were diluted to 1.5 fold their final concentration in assay buffer. The tripeptide substrate and trypsin at 0.05 uM final concentration were diluted in assay buffer at 6 fold their final concentration. The final enzyme concentrations used in these assays were 3.3 ng/ml (HDAC1), 0.2 ng/ml (HDAC2), 0.08 ng/ml (HDAC3) and 2 ng/ml (HDAC6). The final substrate concentrations used were 16 uM (HDAC1), 10 uM (HDAC2), 17 uM (HDAC3) and 14 uM (HDAC6). Five ul of compounds and 20 ul of enzyme were added to wells of a black, opaque 384 well plate in duplicate. Enzyme and compound were incubated together at room temperature for 10 minutes. Five u1 of substrate was added to each well, the plate was shaken for 60 seconds and placed into a Victor 2 microtiter plate reader. The development of fluorescence was monitored for 60 min and the linear rate of the reaction was calculated.
Measurement: IC50 = 8180 nM (pChEMBL 5.090000152587891)
Descriptors: MW 321.38, ALogP 1.04, HBA 6, HBD 3, PSA 107.45, QED 0.56
Compound (SMILES): CNC(=O)C[C@H]1COc2cc(F)ccc2N1C(=O)c1ccc2c(c1)NC(=O)CO2
InChIKey: MBKYLPOPYYLTNW-ZDUSSCGKSA-N
Target: Mineralocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08235
Assay: scintillation proximity assay (SPA): Test A1 and A2: In order to identify binding to the human MR LBD a scintillation proximity assay (SPA) was adapted to the 384-well format. The MR-LBD (amino acids T729-K985) was expressed as N-terminal fusion with maltose binding protein in Hi5 insect cells by co-infection with recombinant MBP-MR LBD and P23 baculoviruses and crude protein lysate was used in the assay. Tritiated aldosterone is used as the ligand to generate the scintillation signal when brought into proximity of the scintillation (SPA) bead by binding to the MR LBD and test compound affinity (in IC50 values) is defined as the concentration to decrease tritiated aldosterone binding to the MR LBD by 50%.Briefly, in Test A1 the assay was run in 384 well format in 10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA 20 mM NaMoO4, 0.1 mM DTT and 10% glycerol at rt. Compounds were tested in a 7 (Test A1) or 10 (Test A2) concentration response curve ranging from 10 nM to 10 μM (Test A1) or 1 nM to 37 μM (Test A2). Compounds were spotted at the bottom of a well of a 384-well PE Opti-Plate to yield final DMSO concentrations in the assay of 2%. Pre-made MBP-MR/P23 lysate: 3H-aldosterone mix (final assay concentration 7 μg/mL MBP-MR LBD/P23 lysate; 5 nM aldosterone) was added onto the top of the spotted compound and preincubated for 1 h at RT. After 1 h an equal volume anti-rabbit SPA beads (Test A1) or Imaging beads (Test A2) coupled with rabbit anti-MBP were added to the assay mixture and incubated for 3 hrs (Test A1) or >8 h (Test A2) at rt. The inhibition of the scintillation signal by displacement of the bound 3H-aldosterone by test compounds is measured by scintillation counting using a Microbeta Trilux (Wallac) (Test A1) or a by CCD camera detection using a LEADseeker (PerkinElmer) (Test A2).
Measurement: IC50 = 37500 nM (pChEMBL 4.429999828338623)
Descriptors: MW 399.38, ALogP 1.7, HBA 5, HBD 2, PSA 96.97, QED 0.82
Compound (SMILES): COc1ccc(CN2CCC(c3ccc4nc(C(=O)N5CCN(Cc6ccc7c(c6)OC(F)(F)O7)CC5)[nH]c4c3)CC2)cn1
InChIKey: MKUPLUBCIIGNHP-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Cell Proliferation Inhibition Assay: In order to make human PIK3 CA mutation-positive breast cancer cell line MDA-MB-453 cells become 500 cells per well in a 384 well non-adhesive plate (Lipidure-Coat plate AT-384; manufactured by NOF Corporation), 36 μl/well was seeded in Leibovitz's L-15 medium including 10% fetal bovine serum (manufactured by Life technologies Corp.), followed by culturing at 37° C. overnight in the presence of CO2. The following day, the test compound (the test compound had 11 steps in a final concentration from 10000 nM to 0.1 nM), and DMSO which is a solvent for the test compound as a negative control were diluted with the medium, and 4 μl was added to cells. Thereafter, the cells were cultured for 4 days at 37° C. in the absence of CO2, and a cytometric reagent (CellTiter-Glo Luminescent Cell Viability assay manufactured by Promega Corporation) was added. The resultant product was stirred for 30 minutes, and then measurement was carried out using a luminescence measurement apparatus
Measurement: IC50 = 8.9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 604.66, ALogP 4.63, HBA 8, HBD 1, PSA 96.05, QED 0.33
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CCC(Nc2cccc(F)c2)CC1
InChIKey: BCAQFASAWFPWRE-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 = 409 nM (pChEMBL 6.389999866485596)
Descriptors: MW 451.48, ALogP 4.06, HBA 7, HBD 3, PSA 121.65, QED 0.47
Compound (SMILES): C[C@@H](c1nc2cccc(Cl)c2c(=O)n1-c1cccc(CN(C)S(C)(=O)=O)c1)N(C)c1ncnc(N)c1C#Cc1ccc(F)cn1
InChIKey: FVYIUOHBSPFKEE-IBGZPJMESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 647.14, ALogP 3.93, HBA 10, HBD 1, PSA 140.2, QED 0.26
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCCC1CN(C(=O)OC(C)(C)C)C(C)(C)CO1
InChIKey: MFHPFBVKYHTFKA-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 577.51, ALogP 6.87, HBA 8, HBD 1, PSA 95.04, QED 0.33
Compound (SMILES): Nc1ncnc(N2CCC(c3nc(-c4ccc(F)c(C(F)(F)F)c4)cn3CCN3CCCC3)CC2)c1-c1ccc(F)cc1
InChIKey: OWCPNEJTTGLSQC-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of human ERG by patch clamp assay
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 597.64, ALogP 6.37, HBA 7, HBD 1, PSA 76.1, QED 0.25
Compound (SMILES): Cc1ccc(C2=C(C#N)C(=O)NC(c3ccc(C(C)(C)C)cc3)(C(F)(F)F)C2)cc1
InChIKey: SWGRXKTWGGZGSR-UHFFFAOYSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: MGAT SPA Assay: MGAT2 enzyme was assayed using membranes isolated from Sf9 cells expressing the recombinant human MGAT2 cDNA with 2-monooleoylglycerol and [3H]-oleoyl-CoA as substrates as described by Seethala et al. [Anal. Biochem., 383(2):144-150 (Dec. 15, 2008)]. Briefly, the assays were conducted in 384-well plates in a total volume of 30 uL at 25° C. In each assay, 200 ng of recombinant human MGAT2 membrane was incubated with 10 uM of 2-monooleoylglycerol and 15 uM of [3H]-oleoyl-CoA in 100 mM potassium phosphate (pH 7.4) for 20 min with various concentrations of compounds delivered in DMSO. The assay was terminated by the addition of 20 ul of Stopping Solution (7.5 mg/ml Yttrium Oxide Polylysine beads, 3.3 mg/ml Fraction V BSA and 200 uM Mercuric chloride in 50 mM HEPES, pH 7.4). The signal was measured 1 h after quenching the reaction using LEADSEEKERSM for 5 minutes. To calculate the degree of inhibition, the zero level of enzyme activity (blank) was defined.
Measurement: IC50 = 564 nM (pChEMBL 6.25)
Descriptors: MW 412.46, ALogP 5.55, HBA 2, HBD 1, PSA 52.89, QED 0.7
Compound (SMILES): CCN(CC)CCN(c1cc(OC)cc(OC)c1)c1ccc2ncc(-c3cnn(C)c3)nc2c1
InChIKey: ZMEWJQRHELGMRY-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 1737.8 nM (pChEMBL 5.760000228881836)
Descriptors: MW 460.58, ALogP 4.53, HBA 8, HBD 0, PSA 68.54, QED 0.34
Compound (SMILES): CO[C@H]1C[C@H](Oc2cc(F)c(F)c(-c3ccc4c(c3)CC[C@H](CCc3nnn[nH]3)O4)c2)C1
InChIKey: IYHNHEJRTXTPSB-BRWVUGGUSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Agonist activity at human GPR40 expressed in HEK293 cells assessed as increase in IP1 accumulation after 60 mins by HTRF assay
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 442.47, ALogP 4.03, HBA 6, HBD 1, PSA 82.15, QED 0.6