lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3c(F)cncc3F)CC2)C1
InChIKey: LVWFVGNNMGWEOB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 503.52, ALogP 2.72, HBA 8, HBD 1, PSA 119.62, QED 0.44
Compound (SMILES): CC1CCCCC1c1nnn2cnc3[nH]ccc3c12
InChIKey: YJOZVKAKNWBYQO-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
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. From the plot of logarithm of a compound concentration and inhibitory activity, the IC50 was calculated.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 255.32, ALogP 2.9, HBA 4, HBD 1, PSA 58.87, QED 0.73
Compound (SMILES): CN1CN([C@H]2CC[C@H](CF)CC2)c2c(cnc3[nH]ccc23)C1=O
InChIKey: VQOZZRKULRLRAA-HAQNSBGRSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 316.38, ALogP 2.94, HBA 3, HBD 1, PSA 52.23, QED 0.93
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3ccc(-c4ccncc4)c(F)c3)CC2)C1
InChIKey: QPVKJTXYRZJLIT-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 561.63, ALogP 4.25, HBA 8, HBD 1, PSA 119.62, QED 0.33
Compound (SMILES): CCn1ccnc1CN(c1cc(OC)cc(OC)c1)c1ccc2ncc(-c3cnn(C)c3)nc2c1
InChIKey: CPKWWDUXGFBILN-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 presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 469.55, ALogP 4.6, HBA 9, HBD 0, PSA 83.12, QED 0.33
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCc1nc(CN(C)C)no1
InChIKey: DBXVNVVGMHBBOC-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
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 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 475.34, ALogP 4.71, HBA 9, HBD 1, PSA 98.43, QED 0.39
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3cccc(OC(F)(F)F)c3)CC2)C1
InChIKey: UNNSPOJJWNTDJL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 550.55, ALogP 3.95, HBA 8, HBD 1, PSA 115.96, QED 0.39
Compound (SMILES): COc1c(Nc2ncc(Cl)c(N[C@H]3[C@@H](C(N)=O)[C@@H]4C=C[C@H]3C4)n2)ccc2c1CCC[C@H](N1CCN(C)CC1)C2
InChIKey: FLTLUXCGVBUQLQ-PDULFCFVSA-N
Target: Focal adhesion kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q05397
Assay: Inhibition of human FAK expressed in baculovirus after 30 mins using biotinyl-amino-hexanoyl-EQEDEPEGDYFEWLE-amide as substrate by TRF assay
Measurement: IC50 = 8.5 nM (pChEMBL 8.069999694824219)
Descriptors: MW 552.12, ALogP 3.46, HBA 8, HBD 3, PSA 108.64, QED 0.35
Compound (SMILES): COc1nn2cc(-c3ccc(OC(F)(F)F)cc3)nc2s1
InChIKey: QYUQSXUEPZTIEG-UHFFFAOYSA-N
Target: Proteinase-activated receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI0
Assay: Antagonist activity at PAR4 in human platelets assessed as inhibition of gamma-thrombin-induced PAC1 binding response preincubated for 20 mins followed by agonist addition for 20 mins by FITC-based flow cytometric analysis
Measurement: IC50 = 229.09 nM (pChEMBL 6.639999866485596)
Descriptors: MW 315.28, ALogP 3.37, HBA 6, HBD 0, PSA 48.65, QED 0.74
Compound (SMILES): Cc1ccc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-c1cc(N2CCOCC2)c(=O)n(C)c1
InChIKey: IINLTCRAWJXNKT-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of CRAF Y340E/Y341E mutant (unknown origin) using human MEK1 K97R mutant as substrate pretreated for 30 mins followed by substrate addition measured after 40 mins by alphascreen assay
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 471.48, ALogP 4.47, HBA 5, HBD 1, PSA 63.57, QED 0.61
Compound (SMILES): Cc1oc2c(c1CC(=O)Nc1nc(-c3ccc(F)c(C(F)(F)F)c3)cs1)c(=O)n(C)c(=O)n2C
InChIKey: ISWKUHZARATATJ-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Radiometric Calcium Assay: The inhibition of TRPA1 receptor activation was measured as inhibition of allylisothiocyanate (AITC) induced cellular uptake of radioactive calcium. Test compounds were dissolved in DMSO to prepare 10 mM stock solution and then diluted using plain medium with 0.1% BSA and 1.8 mM CaCl2 to get desired concentration. Final concentration of DMSO in the reaction was 0.5% (v/v). Human TRPA1 expressing CHO cells were grown in F-12 DMEM medium with 10% FBS, 1% penicillin-streptomycin solution, and 400 ug/ml of G-418. Cells were seeded 24 h prior to the assay in 96 well plates so as to get 50,000 cells per well on the day of experiment. Cells were treated with test compounds for 10 min followed by addition of AITC at a final concentration of 30 micromolar and 5 uCi/ml 45Ca+2 for 3 min at 25° C. Cells were washed and lysed using buffer containing 1% Triton X-100, 0.1% deoxycholate and 0.1% SDS. Radioactivity in the lysate was measured in Packard Top count.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 496.44, ALogP 3.6, HBA 8, HBD 1, PSA 99.13, QED 0.44
Compound (SMILES): CN1C(=O)CCc2cc(-c3cncc4c3CCCC4O)ccc21
InChIKey: JKGUWJPSQLGLQF-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Cellular Enzyme Assay: The expression plasmids contains the ORF for either human/cyno CYP11B1 or CYP11B2 under the control of a suitable promoter (CMV-promoter) and a suitable resistance marker (neomycin). Using standard techniques the expression plasmid is transfected into G-402 cells and these cells are then selected for expressing the given resistance markers. Individual cell-clones are then selected and assessed for displaying the desired enzymatic activity using 11-Deoxycorticosterone (Cyp11B2) or 11-Deoxycortisol (Cyp11B1) as a substrate. G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 μg/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 μM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 206.1 nM (pChEMBL 6.690000057220459)
Descriptors: MW 308.38, ALogP 3.03, HBA 3, HBD 1, PSA 53.43, QED 0.88
Compound (SMILES): COc1cc(CCN2CCOCC2)c(F)cc1Nc1nccc(-c2c(-c3ccc(OC)c(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: JWRDQBNZPRQOCL-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of recombinant human full length ErbB2 expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 709.73, ALogP 6.76, HBA 10, HBD 2, PSA 115.14, QED 0.15
Compound (SMILES): CCOc1cc2ncnc(Nc3ccc(NS(=O)(=O)c4ccccc4)cc3)c2cc1NC(=O)/C=C/CN1CCN(C)CC1
InChIKey: PQMINPFSUDRKEW-JXMROGBWSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Irreversible inhibition of full length human ERBB1 autophosphorylation transfected in EGF-stimulated mouse NIH/3T3 cells incubated for 2 hrs followed by stimulation with EGF for 10 mins
Measurement: IC50 = 390 nM (pChEMBL 6.409999847412109)
Descriptors: MW 601.73, ALogP 4.32, HBA 9, HBD 3, PSA 128.79, QED 0.22
Compound (SMILES): CCCCCC(O)c1ccc([C@@H]2[C@@H](C/C=C\CCCC(=O)O)[C@H](Cl)C[C@H]2Cl)cc1
InChIKey: ZZPVJHFTTJXWMD-IUMCTMNXSA-N
Target: Prostaglandin E2 receptor EP3 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43115
Assay: Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay
Measurement: EC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 441.44, ALogP 6.82, HBA 2, HBD 2, PSA 57.53, QED 0.21
Compound (SMILES): N[C@H](C(=O)O)C(NS(=O)(=O)c1cccnc1)C(=O)O
InChIKey: DKJGSCQSJHEUSX-PKPIPKONSA-N
Target: Excitatory amino acid transporter 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P43004
Assay: Inhibition of [3H]-D-Asp uptake at human EAAT2 expressed in HEK293 cells measured after 3 mins by TopCount method
Measurement: IC50 = 25118.86 nM (pChEMBL 4.599999904632568)
Descriptors: MW 289.27, ALogP -1.77, HBA 6, HBD 4, PSA 159.68, QED 0.48
Compound (SMILES): FC(F)(F)c1cc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)ccc1Cl
InChIKey: QTBXTXTVVCTNQK-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
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 > 1000 nM (pChEMBL 6)
Descriptors: MW 434.85, ALogP 4.66, HBA 5, HBD 1, PSA 62.11, QED 0.51
Compound (SMILES): O=C(CN1CCO[C@H](COc2cccnc2)C1)N1CCc2ccccc21
InChIKey: XYHRHHHLNLGIRG-SFHVURJKSA-N
Target: D(1A) dopamine receptor (SINGLE PROTEIN), Mus musculus
UniProt: Q61616
Assay: Radioligand Binding Assay: The detailed experimental protocols for the radioligand and functional receptor assays are available on the NIMH PDSP website at http://pdsp.med.unc.edu/UNC-CH %20Protocol %20Book.pdf. A. Serotonin receptors: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5A, 5-HT6 and 5-HT7. Assay Buffer: Standard Binding Buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, pH 7.4) Membrane Fraction Source: Transiently or stably transfected cell lines (e.g., HEK293, COS, CHO, NIH3T3). Protocol adapted from Roth et al. (1986), J. Pharmacol. Exp. Ther., 238(2): 480-485; and Roth et al. (1994), J. Pharmacol. Exp. Ther., 268(3): 1403-1410. Adrenergic Receptors: alpha1A, alpha1B, alpha2A, alpha2B, alpha2C, beta1, beta2, beta3 Assay Buffers: For alpha1 receptors, alpha1 Binding Buffer (20 mM Tris-HCl, 145 mM NaCl, pH 7.4); for alpha2 receptors, alpha2 Binding Buffer (50 mM Tris-HCl, 5 mM MgCl2, pH 7.7); for beta receptors.
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 353.42, ALogP 1.75, HBA 5, HBD 0, PSA 54.9, QED 0.82
Compound (SMILES): CC(C)(C)c1cc(NC(=O)Nc2ccc(Oc3ccnc(Nc4ccc5c(cnn5CCN5CCOCC5)c4)n3)c3ccccc23)n(-c2ccc(OCCN3CCOCC3)cc2)n1
InChIKey: CUQPTMGVVQNXDH-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: GSK 3α Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against the GSK 3α enzyme isoform (Invitrogen), are evaluated by determining the level of activation/phosphorylation of the target peptide. The GSK3-α protein (500 ng/mL, 2.5 uL) is mixed with the test compound (2.5 uL at either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL) for 2 hr at RT. The FRET peptide (8 uM, 2.5 uL), which is a phosphorylation target for GSK3α, and ATP (40 uM, 2.5 uL) are then added to the enzyme/compound mixture and the resulting mixture incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 597 nM (pChEMBL 6.21999979019165)
Descriptors: MW 852.01, ALogP 7.69, HBA 14, HBD 3, PSA 157.98, QED 0.1
Compound (SMILES): FC(F)(F)c1cccc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)c1
InChIKey: IABRPRLOJUFYED-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
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 = 4.9 nM (pChEMBL 8.3100004196167)
Descriptors: MW 400.41, ALogP 4, HBA 5, HBD 1, PSA 62.11, QED 0.56
Compound (SMILES): NS(=O)(=O)c1ccc(-n2cc(CSc3ccccc3)nn2)cc1
InChIKey: YCQLKMQECMAFDL-UHFFFAOYSA-N
Target: Carbonic anhydrase 12 (SINGLE PROTEIN), Homo sapiens
UniProt: O43570
Assay: Inhibition of human carbonic anhydrase 12 pre-incubated for 15 mins by stopped flow carbon dioxide hydrase assay
Measurement: Ki = 4.9 nM (pChEMBL 8.3100004196167)
Descriptors: MW 346.44, ALogP 2.21, HBA 6, HBD 1, PSA 90.87, QED 0.72
Compound (SMILES): CCOc1ccc(OC2CCN(c3ncc(C(=O)NO)cn3)CC2)cc1-c1nc2c(CC)nn(C)c2c(=O)[nH]1
InChIKey: GDXUQGMYOAPPJF-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Inhibition of full length recombinant human N-terminal GST tagged HDAC6 expressed in baculovirus infected Sf9 cells using fluorogenic HDAC substrate after 30 mins by fluorescence analysis
Measurement: IC50 = 373 nM (pChEMBL 6.429999828338623)
Descriptors: MW 534.58, ALogP 2.24, HBA 11, HBD 3, PSA 160.38, QED 0.23
Compound (SMILES): Cc1c(C(=O)N2CCc3c(nnn3-c3cnccn3)C2C)cccc1C(F)(F)F
InChIKey: UMGZTIJCJJGATI-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Radioligand Binding assay: Human or rat P2X7-1321N1 cells were collected and frozen @ −80° C. On the day of the experiment, cell membrane preparations were made according to standard published methods. The total assay volume was 100 μl:10 μl compound (10×)+(b) 40 μl tracer (2.5×)+50 μl membrane (2×). The tracer used for the assay was tritiated A-804598. The compound can be prepared as described in the literature. (Donnelly-Roberts, D. Neuropharmacology 2009, 56 (1), 223-229.) Compounds, tracer and membranes were incubated for 1 hour @ 4° C. The assay was terminated by filtration (GF/B filters pre-soaked with 0.3% PEI) and washed with washing buffer (Tris-HCl 50 mM). The IC50 generated in the binding assay was corrected for tracer concentration and affinity of the tracer to derive at the affinity (Ki) of the test compounds.
Measurement: Ki = 2.3 nM (pChEMBL 8.640000343322754)
Descriptors: MW 402.38, ALogP 3.14, HBA 6, HBD 0, PSA 76.8, QED 0.66
Compound (SMILES): COc1cc(OC)cc(N(CCCN2CCC[C@H]2CO)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: LKMLECIRYJPQOC-QFIPXVFZSA-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 presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 502.62, ALogP 4.03, HBA 9, HBD 1, PSA 88.77, QED 0.35
Compound (SMILES): CCc1ccnc(C#CCN(c2cc(OC)cc(OC)c2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)n1
InChIKey: HQQABXMYFDMKIT-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 presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 8511.38 nM (pChEMBL 5.070000171661377)
Descriptors: MW 505.58, ALogP 4.59, HBA 9, HBD 0, PSA 91.08, QED 0.3
Compound (SMILES): Cc1cccc2cc(Cn3nc(-c4ccc5oc(N)nc5c4)c4c(N)ncnc43)n(C3CCC3)c(=O)c12
InChIKey: LTFXZXWIMFYBHO-UHFFFAOYSA-N
Target: PI3K p110 beta/p85 alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P27986
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110beta/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 < 1000 nM (pChEMBL 6)
Descriptors: MW 492.54, ALogP 4.2, HBA 10, HBD 2, PSA 143.67, QED 0.37
Compound (SMILES): CCN1CN([C@H]2CC[C@H](CNCC(F)(F)F)CC2)c2c(cnc3[nH]ccc23)C1=O
InChIKey: GEAYPTLMWXECFV-HDJSIYSDSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 260 nM (pChEMBL 6.579999923706055)
Descriptors: MW 409.46, ALogP 3.51, HBA 4, HBD 2, PSA 64.26, QED 0.79
Compound (SMILES): Cc1ccc(C(=O)N2[C@H](Cc3cccc(-n4nccn4)c3)COC[C@@H]2C)c(-n2nccn2)c1
InChIKey: FCVGUKJNRFNLDS-GHTZIAJQSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Antagonist activity at human OX2R expressed in CHO cells preincubated for 120 mins followed by orexin-A addition by FLIPR assay
Measurement: IC50 = 1827 nM (pChEMBL 5.739999771118164)
Descriptors: MW 443.51, ALogP 2.63, HBA 8, HBD 0, PSA 90.96, QED 0.47
Compound (SMILES): CCOc1ccc(C2CCC(CC(=O)NO)CC2)cc1-c1nc2c(CC)nn(C)c2c(=O)[nH]1
InChIKey: NSKSQVVODLRDIX-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Inhibition of full length recombinant human N-terminal GST tagged HDAC6 expressed in baculovirus infected Sf9 cells using fluorogenic HDAC substrate after 30 mins by fluorescence analysis
Measurement: IC50 = 5130 nM (pChEMBL 5.289999961853027)
Descriptors: MW 453.54, ALogP 3.45, HBA 7, HBD 3, PSA 122.13, QED 0.37
Compound (SMILES): CO[C@H]1C[C@@H](n2c(=O)n(C)c3cnc4ccc(-c5ccc(OCCCN(C)C)nc5)cc4c32)C1
InChIKey: DWICJHMMZLNUEB-BGYRXZFFSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Cellular Assay: Assay d) MDA-MB-468 cells (human breast adenocarcinoma ##ATCC HTB 132) were seeded at 1500 cells/well in 40 μl of DMEM containing 10% FBS and 1% glutamine into Greiner 384 well black flat-bottomed plates. Cell plates were incubated for 18 h in a 37° C. incubator before dosing with compounds of formula (I) in 100% DMSO using acoustic dispensing. Compounds were dosed in a 12 point concentration range into a randomised plate map. Control wells were generated either by dosing of 100% DMSO (max signal) or addition of a reference compound (a PI3K-β inhibitor) that completely eliminated the pAKT signal (min control). Plates were incubated, at 37° C. for 2 h, cells were then fixed by the addition of 10 μl of a 3.7% formaldehyde solution. After 30 minutes the plates were washed with PBS using a Tecan PW384 plate washer. Wells were blocked and cells permeabilised with the addition of 40 μl of PBS containing 0.5% Tween20 and 1% Marvel™ (dried milk powder) and incubated for 60 minutes at r.t. The plates were washed with PBS containing 0.5% (v/v) Tween20 and 20 μl rabbit anti-phospho AKT Ser473 (Cell Signalling Technologies, #3787) in same PBS-Tween+1% Marvel™ was added and incubated overnight at 4° C. Plates were washed 3 times with PBS 0.05% Tween 20 using a Tecan PW384. 20 μl of secondary antibody Alexa Fluor 488 anti-Rabbit (Molecular Probes, #A11008) diluted in PBS+0.05% Tween20 containing 1% Marvel™ was added to each well and incubated for 1 h at r.t. Plates were washed three times as before then 20 μl PBS added to each well and plates sealed with a black plate sealer. The plates were read on an Acumen plate reader as soon as possible, measuring green fluorescence after excitation with 488 nm laser.
Measurement: IC50 = 1170 nM (pChEMBL 5.929999828338623)
Descriptors: MW 461.57, ALogP 3.63, HBA 8, HBD 0, PSA 74.41, QED 0.37
Compound (SMILES): C[C@@H]1CN(c2nnc(Cc3ccc(F)cc3)c3c2CCC3)CCN1c1cc(C(F)(F)F)c(C(C)(C)O)cn1
InChIKey: QQXLSYYOFWNBRB-QGZVFWFLSA-N
Target: Protein smoothened (SINGLE PROTEIN), Homo sapiens
UniProt: Q99835
Assay: Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 529.58, ALogP 5.05, HBA 6, HBD 1, PSA 65.38, QED 0.47
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1Cl)N1CCc2c(nnn2-c2ccccc2)C1
InChIKey: SQRLNRNKCKCMFY-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Ca2+ flux assay: 1321N1 cells expressing the recombinant human or rat 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 μl 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 250× the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 μL of the compound into 300 μL of assay buffer. A further 3× dilution occurred when transferring 50 μL/well of the compound plate to 100 μL/well in the cell plate. Cells were incubated with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 μL/well of BzATP (final concentration is 250 μM BzATP (human and rat)). The fluorescence change was measured 180 seconds after adding the agonist. Peak fluorescence was plotted as a function of BzATP concentration using Origin 7 software.
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 406.8, ALogP 4.14, HBA 4, HBD 0, PSA 51.02, QED 0.64
Compound (SMILES): C=CC(=O)N1CCC([C@@H]2CCNc3c(C(N)=O)c(-c4ccc(Oc5ccccc5)cc4)nn32)CC1
InChIKey: RNOAOAWBMHREKO-QFIPXVFZSA-N
Target: Cytoplasmic tyrosine-protein kinase BMX (SINGLE PROTEIN), Homo sapiens
UniProt: P51813
Assay: Inhibition of full-length N-terminal GST-tagged BMX (1 to 675 residues) (unknown origin) expressed in Sf21 insect cells using NH2-ETVYSEVRK-biotin as substrate preincubated for 1 hr followed by ATP addition and measured after 2 hrs by ELISA
Measurement: IC50 = 5.4 nM (pChEMBL 8.270000457763672)
Descriptors: MW 471.56, ALogP 4.22, HBA 6, HBD 2, PSA 102.48, QED 0.52
Compound (SMILES): CNc1cccc(N(CC2CC2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: VCHBCVRCEHYVAE-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 presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 384.49, ALogP 4.62, HBA 6, HBD 1, PSA 58.87, QED 0.53
Compound (SMILES): Cc1ncoc1-c1nnc(SCCN2CC[C@]3(C[C@@H]3c3ccc(C(F)(F)F)cc3)C2)n1C
InChIKey: KLQDZSYTMPJBBU-UTKZUKDTSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Antagonist activity at human dopamine D3 receptor expressed in CHO cell membranes after 90 mins in presence of quinelorane by [35S]-GTPgammaS binding assay
Measurement: Ki = 53.7 nM (pChEMBL 7.269999980926514)
Descriptors: MW 463.53, ALogP 4.77, HBA 7, HBD 0, PSA 59.98, QED 0.49
Compound (SMILES): COc1cc(N2CCC(N3CCC(O)CC3)CC2)ccc1Nc1nccc(-c2c(-c3ccc(OC)c(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: MRHINXBPDMMURR-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of recombinant human full length ErbB2 expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 760.85, ALogP 7.18, HBA 11, HBD 3, PSA 129.38, QED 0.13
Compound (SMILES): COc1cc(OC)cc(N(CC(O)CN)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: JRZBGTIYYHVJEO-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 presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 434.5, ALogP 2.51, HBA 9, HBD 2, PSA 111.55, QED 0.44
Compound (SMILES): N#CCC1(n2cc(-c3c(C#N)cnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3cc(C#N)cc(C#N)c3)CC2)C1
InChIKey: AOWQKMUADIXHEC-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 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 540.59, ALogP 3.27, HBA 9, HBD 1, PSA 165.21, QED 0.4
Compound (SMILES): Cn1cc(Cn2cc(-c3ccc(Cl)c(OC(F)F)c3)cn2)cn1
InChIKey: TYWLMLPPMXWFPX-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of Specific Binding to the hERG-Receptor (HERGBD): After thawing on the day of the assay, the membrane suspension was diluted further with 20 mM HEPES/0.1 mM KCl/pH 7.2.The incubation mixture of 200 μl contained 1.5 nmol/l 3H-Dofetilide, optimized amount of membrane preparation, 20 mM HEPES/0.1 mM KCl/(pH 7.2) and inhibitor in 1% DMSO. Nonspecific binding was estimated in the presence of 10 M Dofetilide. The samples were incubated for 90 min. at RT.Binding was terminated by filtration of the incubated membrane preparations using Filtermat B (Pharmacia, Uppsala Sweden) and a Micro Cell Harvester (Skatron, Lier, Norway). The Filtermat B had been presoaked with 1% polyethylen imine and carefully washed with 0.05 M Tris/HCl-buffer pH=7.7 after the filtration to separate free and bound radioactivity. The filters were counted in a scintillation counter (Betaplate 1205, Berthold, Wildbad, Germany) in order to determine the specific binding of [3H]-Dofetilide.
Measurement: IC50 = 119 nM (pChEMBL 6.920000076293945)
Descriptors: MW 338.75, ALogP 3.59, HBA 5, HBD 0, PSA 44.87, QED 0.71
Compound (SMILES): Cc1c(C(=O)N2Cc3nnn(-c4ncc(F)cn4)c3C[C@@H]2C)ccnc1C(F)(F)F
InChIKey: VYDQRUVOXRXKGT-VIFPVBQESA-N
Target: Cytochrome P450 2C9 (SINGLE PROTEIN), Homo sapiens
UniProt: P11712
Assay: Inhibition of CYP2C9 in human liver microsomes using tolbutamide or diclofenac as substrates
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 421.36, ALogP 2.51, HBA 7, HBD 0, PSA 89.69, QED 0.59
Compound (SMILES): Cc1nsc(-c2nnc3n2CCN(C(=O)c2ccc(Cl)c(Cl)c2)C3C)n1
InChIKey: ZMVUVIKWZWFBIB-UHFFFAOYSA-N
Target: Substance-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21452
Assay: Binding Assay: The affinity of compounds of the invention for the NK-2 receptor was evaluated in CHO recombinant cells which express the human NK-2 receptor. Membrane suspensions were prepared from these cells. The following radioligand [125I]-Neurokinin A (PerkinElmer Cat#NEX252) was used in this assay. Binding assays were performed in a 25 mM HEPES/1 mM CaCl2/5 mM MgCl2/0.5% BSA/10 μg/ml saponin, at pH 7.4. Binding assays consisted of 25 μl of membrane suspension (approximately 3.75 μg of protein/well in a 96 well plate), 50 μl of compound or reference ligand (Neurokinin A) at increasing concentrations (diluted in assay buffer) and 0.1 nM [125I]-Neurokinin A. The plate was incubated 60 min at 25° C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in assay buffer without saponine for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard).
Measurement: Ki = 28000 nM (pChEMBL 4.550000190734863)
Descriptors: MW 409.3, ALogP 3.63, HBA 7, HBD 0, PSA 76.8, QED 0.65
Compound (SMILES): Clc1ccc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)c(Cl)n1
InChIKey: LBBCZEDNOMVQRU-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
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 = 610 nM (pChEMBL 6.210000038146973)
Descriptors: MW 402.29, ALogP 3.69, HBA 6, HBD 1, PSA 75, QED 0.53
Compound (SMILES): CC(C)c1cccc(C(C)C)c1NC(=O)CC(=O)C(Cc1ccccc1)Cc1ccc(OC(=O)CN2CCCCCC2=O)cc1
InChIKey: NCYIHYBRISFJLW-UHFFFAOYSA-N
Target: Sterol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75908
Assay: Inhibition Assay: Methods for assessing the selectively of ACAT1 inhibitors are known in the art and can be based upon any conventional assay including, but not limited to the determination of the half maximal (50%) inhibitory concentration (IC) of a substance (i.e., 50% IC, or IC50), the binding affinity of the inhibitor (i.e., Ki), and/or the half maximal effective concentration (EC50) of the inhibitor for ACAT1 as compared to ACAT2. See, e.g., Lada, et al. (2004) J. Lipid Res. 45:378-386 and U.S. Pat. No. 5,968,749. ACAT1 and ACAT2 proteins that can be employed in such assays are well-known in the art and set forth, e.g., in GENBANK Accession Nos. NP-000010 (human ACAT1) and NP-005882 (human ACAT2). See also U.S. Pat. No. 5,834,283.
Measurement: IC50 = 600 nM (pChEMBL 6.21999979019165)
Descriptors: MW 610.8, ALogP 7.24, HBA 5, HBD 1, PSA 92.78, QED 0.13
Compound (SMILES): Cc1ccc(C(=O)N2CCOC[C@H]2Cc2cc(-n3nccn3)ccc2Cl)c(-n2nccn2)c1
InChIKey: PHEQMGRTEIENAJ-LJQANCHMSA-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 = 19000 nM (pChEMBL 4.71999979019165)
Descriptors: MW 463.93, ALogP 2.89, HBA 8, HBD 0, PSA 90.96, QED 0.45
Compound (SMILES): CCN(CC)c1ccc(-c2cc(-c3ccc(C(=O)O)cc3)ccc2OCCO)cc1C(C)(C)C
InChIKey: OOTHJAQLAQMOBN-UHFFFAOYSA-N
Target: Retinoic acid receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P13631
Assay: Agonist activity at GAL4 DNA-binding domain-tagged RARgamma (unknown origin) ligand-binding domain expressed in human HG5LN cells incubated for 18 hrs by luciferase reporter gene assay
Measurement: EC50 = 3.6 nM (pChEMBL 8.4399995803833)
Descriptors: MW 461.6, ALogP 6.23, HBA 4, HBD 2, PSA 70, QED 0.4
Compound (SMILES): CN1CCC(c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: SOVFLDCVIVRQKC-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
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 > 1000 nM (pChEMBL 6)
Descriptors: MW 256.31, ALogP 1.41, HBA 5, HBD 1, PSA 62.11, QED 0.71
Compound (SMILES): CC(C)(C)c1cc(NC(=O)Nc2ccc(Oc3ccnc(Nc4ccc5c(c4)NC(=O)C5)n3)c3ccccc23)n(-c2cccc(OCCN3CCOCC3)c2)n1
InChIKey: SATISEDUDGLGFE-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: GSK 3α Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against the GSK 3α enzyme isoform (Invitrogen), are evaluated by determining the level of activation/phosphorylation of the target peptide. The GSK3-α protein (500 ng/mL, 2.5 uL) is mixed with the test compound (2.5 uL at either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL) for 2 hr at RT. The FRET peptide (8 uM, 2.5 uL), which is a phosphorylation target for GSK3α, and ATP (40 uM, 2.5 uL) are then added to the enzyme/compound mixture and the resulting mixture incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 1170 nM (pChEMBL 5.929999828338623)
Descriptors: MW 753.86, ALogP 7.5, HBA 11, HBD 4, PSA 156.79, QED 0.1
Compound (SMILES): CO[C@H]1C[C@@H](n2c(=O)n(C)c3cnc4ccc(-c5ccc(OCCCN6CCCC6)nc5)cc4c32)C1
InChIKey: OPNZWUZVFPAJIO-SZPZYZBQSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Cellular Assay: Assay d) MDA-MB-468 cells (human breast adenocarcinoma ##ATCC HTB 132) were seeded at 1500 cells/well in 40 μl of DMEM containing 10% FBS and 1% glutamine into Greiner 384 well black flat-bottomed plates. Cell plates were incubated for 18 h in a 37° C. incubator before dosing with compounds of formula (I) in 100% DMSO using acoustic dispensing. Compounds were dosed in a 12 point concentration range into a randomised plate map. Control wells were generated either by dosing of 100% DMSO (max signal) or addition of a reference compound (a PI3K-β inhibitor) that completely eliminated the pAKT signal (min control). Plates were incubated, at 37° C. for 2 h, cells were then fixed by the addition of 10 μl of a 3.7% formaldehyde solution. After 30 minutes the plates were washed with PBS using a Tecan PW384 plate washer. Wells were blocked and cells permeabilised with the addition of 40 μl of PBS containing 0.5% Tween20 and 1% Marvel™ (dried milk powder) and incubated for 60 minutes at r.t. The plates were washed with PBS containing 0.5% (v/v) Tween20 and 20 μl rabbit anti-phospho AKT Ser473 (Cell Signalling Technologies, #3787) in same PBS-Tween+1% Marvel™ was added and incubated overnight at 4° C. Plates were washed 3 times with PBS 0.05% Tween 20 using a Tecan PW384. 20 μl of secondary antibody Alexa Fluor 488 anti-Rabbit (Molecular Probes, #A11008) diluted in PBS+0.05% Tween20 containing 1% Marvel™ was added to each well and incubated for 1 h at r.t. Plates were washed three times as before then 20 μl PBS added to each well and plates sealed with a black plate sealer. The plates were read on an Acumen plate reader as soon as possible, measuring green fluorescence after excitation with 488 nm laser.
Measurement: IC50 = 2770 nM (pChEMBL 5.559999942779541)
Descriptors: MW 487.6, ALogP 4.16, HBA 8, HBD 0, PSA 74.41, QED 0.35
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(C#CC3CC3)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: BVZRDCPNICTPGQ-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 460.54, ALogP 4.14, HBA 7, HBD 1, PSA 91.62, QED 0.41