lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): FC1(F)Oc2ccc(CN3CCC(c4nnn5cnc6[nH]ccc6c45)CC3)cc2O1
InChIKey: IBELNCZALJQGIS-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 = 79 nM (pChEMBL 7.099999904632568)
Descriptors: MW 412.4, ALogP 3.31, HBA 7, HBD 1, PSA 80.57, QED 0.56
Compound (SMILES): Cc1c(S(=O)(=O)N2CCC(Oc3ccc(Cl)cc3Cl)CC2)c(C(F)(F)F)nn1C
InChIKey: NNKLXAPKUATLRT-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Biological Assay: The ability of a compound to block the intracellular release of calcium mediated by PK1 in RBL2H3 cells expressing human PKR1 receptors is determined as a measure of the compound's antagonist activity in vitro.Approximately 10,000 cells per assay well are seeded in normal culture medium in a 384 well plate (Corning). Twenty-four hours after seeding, the cells are loaded with a calcium sensitive fluorescent dye by replacing the culture medium with assay buffer (1× Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH 7.4) containing 1 mM probenecid and 1× Calcium 5 Reagent (Molecular Devices). Cells are incubated at 37° C. for 1 hour to allow for dye uptake.To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 μM (diluted in assay buffer) are added to the assay wells and allowed to incubate for 10 minutes prior to stimulation with PK1. After incubation with test compounds the assay plate is placed in a FLIPR Tetra (Molecular Devices) and PK1 (diluted in assay buffer) is added at the determined EC80 concentration (final). Ligand-dependent changes in intracellular calcium levels are determined by measuring changes in fluorescence of the dye at 525 nM following to excitation at 485 nM.
Measurement: IC50 = 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 472.32, ALogP 4.29, HBA 5, HBD 0, PSA 64.43, QED 0.67
Compound (SMILES): CCCS(=O)(=O)Nc1ccc(F)c(C(=O)c2c[nH]c3ncc(-c4ccc(F)nc4)cc23)c1F
InChIKey: UBJUBKFKARGYJS-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of C-Raf1 (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 474.46, ALogP 4.42, HBA 5, HBD 2, PSA 104.81, QED 0.3
Compound (SMILES): O=C(CCCCCn1cc(-c2ccc3ncnc(Nc4ccc(OCc5cccc(F)c5)c(Cl)c4)c3c2)nn1)NO
InChIKey: JDBWHHHWEABNEC-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of HER2 (unknown origin) assessed as remaining ATP level measured after 15 mins by luminescence analysis
Measurement: IC50 = 192.8 nM (pChEMBL 6.710000038146973)
Descriptors: MW 576.03, ALogP 6.07, HBA 9, HBD 3, PSA 127.08, QED 0.09
Compound (SMILES): O=C(CO)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(O)c1)C2)Cc1c(F)cccc1F
InChIKey: HIZQWWSJSYDBOP-REPLKXPHSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Displacement of [3H]-diprenorphine from human mu opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 430.5, ALogP 3.4, HBA 4, HBD 2, PSA 64.01, QED 0.71
Compound (SMILES): COc1cc(OC)cc(N(CC#Cc2ccnc(N)n2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: DBFMFHYDGXJVLJ-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 = 2238.72 nM (pChEMBL 5.650000095367432)
Descriptors: MW 492.54, ALogP 3.61, HBA 10, HBD 1, PSA 117.1, QED 0.35
Compound (SMILES): CN(C)c1ccc2cccc(OCC(=O)NC[C@H](O)CN3CCc4ccccc4C3)c2n1
InChIKey: NQYRQXHXODTULO-NRFANRHFSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Biochemical Assay: Molecular Biology: Full-length human PRMT5 (NM_006109.3) transcript variant 1 clone was amplified from a fetal brain cDNA library, incorporating flanking 5′ sequence encoding a FLAG tag (MDYKDDDDK) (SEQ ID NO.:4) fused directly to Ala 2 of PRMT5. Full-length human MEP50 (NM_024102) clone was amplified from a human testis cDNA library incorporating a 5′ sequence encoding a 6-histidine tag (MHHHHHH) (SEQ ID NO.:5) fused directly to Arg 2 of MEP50. The amplified genes were sublconed into pENTR/D/TEV (Life Technologies) and subsequently transferred by Gateway attL×attR recombination to pDEST8 baculvirus expression vector (Life Technologies).Protein Expression. Recombinant baculovirus and Baculovirus-Infected Insect Cells (BIIC) were generated according to Bac-to-Bac kit instructions (Life Technologies) and Wasilko, 2006, respectively. Protein over-expression was accomplished by infecting exponentially growing Spodoptera frugiperda (SF9) cell culture at 1.2×106 cell/ml with a 5000 fold dilution of BIIC stock. Infections were carried out at 27° C. for 72 hours, harvested by centrifugation, and stored at −80° C. for purification.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 434.54, ALogP 2.21, HBA 6, HBD 2, PSA 77.93, QED 0.57
Compound (SMILES): Nc1ncnc2c1c(-c1ccc(Br)c(O)c1)nn2C1Cc2ccccc2C1
InChIKey: FZTMVHAUQSSXMI-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Inhibition of PI3Kdelta (unknown origin)
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 422.29, ALogP 3.88, HBA 6, HBD 2, PSA 89.85, QED 0.51
Compound (SMILES): Cc1cccc(-c2ocnc2C(=O)Nc2cnn(CCOc3cnc4ccccc4n3)c2)c1
InChIKey: PHLDAHHMNAPBQI-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Antagonist activity at human OX1R expressed in CHO cells assessed as orexin A-induced intracellular Ca2+ mobilization preincubated for 120 mins followed by orexin A addition by FLIPR assay
Measurement: IC50 = 1351 nM (pChEMBL 5.869999885559082)
Descriptors: MW 440.46, ALogP 4.12, HBA 8, HBD 1, PSA 107.96, QED 0.41
Compound (SMILES): NC(=O)c1cccc([C@H]2C[C@H]3CC[C@@H](C2)N3CCN(Cc2ccccc2)C(=O)[C@@H](O)CO)c1.O=C(O)C(F)(F)F
InChIKey: ZPLRMXXTPUHPSX-WTKDUOFSSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Displacement of [3H]-diprenorphine from human mu opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 451.57, ALogP 1.88, HBA 5, HBD 3, PSA 107.1, QED 0.54
Compound (SMILES): COc1cc(O)cc(N(CCNC(C)C)c2ccc3ncc(-c4cn[nH]c4)nc3c2)c1.Cl
InChIKey: WDACLZKYOGTKNW-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 418.5, ALogP 3.87, HBA 7, HBD 3, PSA 99.19, QED 0.4
Compound (SMILES): Cc1cccc(-c2cc(N)nc3[nH]cc(C#N)c23)c1
InChIKey: KWTGXMXCSXFSJX-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Inhibition of wild type recombinant human GST-tagged LRRK2 (970 to 2527 residues) expressed in baculovirus using fluorescein-LRRKtide as substrate after 2 hrs by TR-FRET assay
Measurement: Ki = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 248.29, ALogP 2.99, HBA 3, HBD 2, PSA 78.49, QED 0.69
Compound (SMILES): CCCS(=O)(=O)Nc1ccc(F)c(C(=O)c2c[nH]c3ncc(-c4ccc(O)nc4)cc23)c1F
InChIKey: MKVMMAUBKKDDSE-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of C-Raf1 (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 472.47, ALogP 3.99, HBA 6, HBD 3, PSA 125.04, QED 0.35
Compound (SMILES): Cc1cc(Nc2nccc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)nn4-c4ccc(=O)n(CCN5CCOCC5)c4)c4ccccc34)n2)cc2cn[nH]c12
InChIKey: XIFFATZVUNRCKL-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 = 2780 nM (pChEMBL 5.559999942779541)
Descriptors: MW 753.87, ALogP 6.97, HBA 12, HBD 4, PSA 169.14, QED 0.11
Compound (SMILES): CN(C)CCOc1ccc(/C(=C(/CCCF)c2ccccc2)c2ccc(O)cc2)cc1.Cl
InChIKey: KXTIGEUSLGAAHZ-JTHROIFXSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: Inhibition of fluorescien-conjugated coactivator PGC1a binding to ERalpha (unknown origin) after 1 hr by TR-FRET assay
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 419.54, ALogP 6.04, HBA 3, HBD 1, PSA 32.7, QED 0.41
Compound (SMILES): CN(C)CCCOc1ccc(-c2ccc3ncc4c(c3c2)n(C2(C)CCOCC2)c(=O)n4C)cn1
InChIKey: SIKMYPMDQGCEPL-UHFFFAOYSA-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 = 266 nM (pChEMBL 6.579999923706055)
Descriptors: MW 475.59, ALogP 3.81, HBA 8, HBD 0, PSA 74.41, QED 0.38
Compound (SMILES): O=C(OC(C(F)(F)F)C(F)(F)F)N1CCN(C(c2ccc(Cl)cc2)c2ccc(Cl)cc2)CC1
InChIKey: UPIHQFKOCOKGEO-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: Inhibition of MGLL derived from human PC3 cell lysates preincubated for 30 mins followed by JW912 addition after 30 mins by gel-based ABPP assay
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 515.28, ALogP 6.33, HBA 3, HBD 0, PSA 32.78, QED 0.45
Compound (SMILES): CNCCN1CCC(OCc2ccccc2)CC1
InChIKey: HBOJWAYLSJLULG-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96LA8
Assay: Binding affinity to human PRMT6 in presence of SAH by ITC method
Measurement: Kd = 87 nM (pChEMBL 7.059999942779541)
Descriptors: MW 248.37, ALogP 1.89, HBA 3, HBD 1, PSA 24.5, QED 0.83
Compound (SMILES): O=C(NO)c1cnc(NC2(c3ccccc3)CCN(C(=O)Nc3ccc(F)cc3)CC2)nc1
InChIKey: OTLJTZYWUNDMTL-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 450.47, ALogP 3.37, HBA 6, HBD 4, PSA 119.48, QED 0.35
Compound (SMILES): COc1cc(N2CCC(C3CCN(CCS(C)(=O)=O)CC3)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: YZEQLRRLTUYDIQ-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 < 100 nM (pChEMBL 7)
Descriptors: MW 820.97, ALogP 7.71, HBA 11, HBD 2, PSA 134.06, QED 0.13
Compound (SMILES): COc1c(Nc2ncc(Cl)c(Nc3ccccc3N(C)S(C)(=O)=O)n2)ccc2c1CCCC(N1CCN(C)CC1)C2
InChIKey: ZTOIRWCKMDSZQZ-UHFFFAOYSA-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 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 600.19, ALogP 4.52, HBA 9, HBD 2, PSA 102.93, QED 0.36
Compound (SMILES): COc1nc(N(CC(C)(C)F)c2cc(Cl)c(F)cc2F)cnc1C(=O)[C@H]1C[C@@H]1C(=O)O
InChIKey: RVMWIVNHZMXEHS-UWVGGRQHSA-N
Target: Cytochrome P450 2C9 (SINGLE PROTEIN), Homo sapiens
UniProt: P11712
Assay: Inhibition of recombinant human CYP2C9 expressed in Escherichia coli using coumarin based substrate by fluorescence assay
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 457.84, ALogP 4.21, HBA 6, HBD 1, PSA 92.62, QED 0.47
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4nc(C)n(Cc5ccc(F)cc5)c(=O)c4c3)cc12
InChIKey: VMEBZEMJNJSXPG-UHFFFAOYSA-N
Target: RNA-directed RNA polymerase (SINGLE PROTEIN), Hepacivirus hominis
UniProt: O39930
Assay: Non-nucleoside inhibition of NS5B polymerase in HCV genotype 2b by Taqman probe based RT-PCR analysis
Measurement: EC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 642.68, ALogP 5.87, HBA 7, HBD 1, PSA 114.51, QED 0.23
Compound (SMILES): CN(c1cccc(C#N)n1)[C@@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: DKFDJOXMHHYUSX-CYBMUJFWSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzymatic Assay: JAK1/2/3 kinase assay are performed in vitro using Kit-Tyr 6 Peptide (Invitrogen, Cat. No. PV4122). TYK2 kinase assay are performed in vitro using Z′-LYTE Kinase Assay Kit-Tyr 3 Peptide (Invitrogen, Cat. No. PV3192). Recombinant human JAK1/2/3 or TYK2 catalytic domains are from Invitrogen (Cat No. PV4774/PV4210/PV3855/PV4790); All reactions (20 μL) are started by adding 2.5 μL of the testing compound in 4% DMSO solution, 5 μL of Kinase/Peptide substrate Mixture (3.2, 0.04, 0.2 or 8 μg/mL for Recombinant human JAK1/2/3 catalytic domains, 4 μM for Z-LYTE Tyr 6 peptide or Z-LYTE Tyr 3 peptide) or Phospho-Peptide solution (Invitrogen, Cat. No. PV3192, diluted with 1.33× Kinase Buffer), 2.5 μL ATP Solution (300/100/40/100 μM, JAK1/JAK2/JAK3/TYK2) or 1.33× Kinase Buffer (Invitrogen, Cat. No. PV3189, 5× diluted with distilled water). The 384-well assay plate (Corning, Cat. No. 3575) is mixed and incubated at room temperature for 1 hour. 5 μL of the Development Solution (Dilute Development Reagent A (Cat. No. PV3297) is diluted to 1/64 with Development Buffer (Cat. No. PV3127) for JAK1, JAK2 and JAK3 assay; Development Reagent A (Cat. No. PV3297) is diluted to 1/2048 with Development Buffer (Cat. No. PV3127) for TYK2 assay. The diluted Development Solution is then added to each well, mixed and incubated at room temperature for another 1 hour. The kinase reaction is then stopped by adding 5 μL of the Stop Reagent (Invitrogen, Cat. No. PV3094), and the plate is read with Wallac 1420 VICTOR3 Multilabel Counter (PerkinElmer) at 445 nm and 520 nm fluorescence. All compounds are initially tested at 8 different concentrations (1 μM down to 0.0003 μM) using a 1:3 serial dilution scheme.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 319.37, ALogP 1.94, HBA 6, HBD 1, PSA 84.73, QED 0.79
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)nn2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3CC4)cc12
InChIKey: FGNRMZOEKSHEBT-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1b (isolate Japanese) (HCV)
UniProt: P26662
Assay: Inhibition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 8.465 nM (pChEMBL 8.069999694824219)
Descriptors: MW 612.66, ALogP 5.27, HBA 7, HBD 1, PSA 101.6, QED 0.29
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2[C@@H]3CC[C@H]2[C@H](COc2ccc(F)cn2)C3)n1
InChIKey: WGJDXEJAXMDTDU-UAGQMJEPSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Binding affinity to human OX2R
Measurement: Ki = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 408.44, ALogP 2.58, HBA 7, HBD 0, PSA 86.03, QED 0.65
Compound (SMILES): COc1cc(OC)cc(N(Cc2csc(C)n2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: IIGRWRUGUUITMA-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 472.57, ALogP 5.15, HBA 9, HBD 0, PSA 78.19, QED 0.32
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(OCC3CCC3)cc1C21N=C(C)C(N)=N1
InChIKey: XWVPZETUJYENCA-VYNBAUGJSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Diluted TR-FRET Assay: TR-FRET Assay: The 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 50 mM Glycine pH 2.5, adjusted to pH 7.4 with 1 M Tris and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the TruPoint BACE1 Substrate to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). Enzyme and compound in dimethylsulphoxide (final DMSO concentration 5%) was mixed and pre-incubated for 10 minutes at RT. Substrate was then added and the reaction was incubated for 15 minutes at RT. The reaction was stopped with the addition of 0.35 vol Stop solution (NaAcetate, pH 9).
Measurement: IC50 = 6.1 nM (pChEMBL 8.210000038146973)
Descriptors: MW 381.52, ALogP 3.98, HBA 5, HBD 1, PSA 69.2, QED 0.86
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)OCC3CC3)CC2)C1
InChIKey: HWFLJCHMMMZJHB-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 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 460.54, ALogP 2.76, HBA 8, HBD 1, PSA 115.96, QED 0.6
Compound (SMILES): COc1cccc(-c2nn(Cc3cc4cccc(C)c4c(=O)n3-c3ccccc3C)c3ncnc(N)c23)c1
InChIKey: ZKCNTUKOSVMBDG-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 < 10000 nM (pChEMBL 5)
Descriptors: MW 502.58, ALogP 5.05, HBA 8, HBD 1, PSA 100.85, QED 0.35
Compound (SMILES): COc1cc(N2CCN(CCS(C)(=O)=O)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: ACEREWSFULKZGV-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 < 100 nM (pChEMBL 7)
Descriptors: MW 738.82, ALogP 5.91, HBA 11, HBD 2, PSA 134.06, QED 0.17
Compound (SMILES): COc1cc(OC)cc(N(CCN2CC[C@H](C#N)C2)c2ccc3ncc(-c4cnn(CC5CCOCC5)c4)nc3c2)c1
InChIKey: FNJXQSFBTXMNDB-XMMPIXPASA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Inhibition of FLT3 (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 567.69, ALogP 4.92, HBA 10, HBD 0, PSA 101.56, QED 0.26
Compound (SMILES): CCN(CC)CCNC(=O)c1cc2nccc(Oc3ccc(NC(=O)Nc4cc(C)ccc4F)cc3)c2s1
InChIKey: YCZNRSBAKIVMMQ-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P17948
Assay: FLIPR (Fluorometric Imaging Plate Reader) Technology Assay: Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of PDGF induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. NHDF-Ad (Normal human dermal fibroblasts) (Lonza) were seeded in 384-well fibronectin coated black-walled plates overnight at 37° C./5% CO2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37° C. Cells were washed 4 times (ELx405-CW, Bio-Tek) to remove extracellular dye. Test compounds were reconstituted in 100% DMSO and added to the cells to give a final DMSO concentration of 0.1%. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 μM) or at concentrations ranging from 0.001 nM to 10 μM followed by PDGF stimulation (10 ng/mL). Changes in fluorescence at 515 nm were measured simultaneously in all 384 wells using a cooled CCD camera.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 535.65, ALogP 6.25, HBA 6, HBD 3, PSA 95.59, QED 0.22
Compound (SMILES): CC1c2ncn(-c3ccc(F)cn3)c2CCN1C(=O)c1cccc(C(F)(F)F)c1F
InChIKey: AKARXLACIWHCCM-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
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 = 3467 nM (pChEMBL 5.460000038146973)
Descriptors: MW 422.36, ALogP 4.32, HBA 4, HBD 0, PSA 51.02, QED 0.58
Compound (SMILES): CC(=O)c1cccc(C#Cc2c(N)ncnc2N[C@@H](C)c2nc3cccc(Cl)c3c(=O)n2-c2ccccc2)c1
InChIKey: ZVVCFSFQXLPNDM-SFHVURJKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
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 535.01, ALogP 5.19, HBA 8, HBD 2, PSA 115.79, QED 0.24
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)C3CCC(F)(F)CC3)CC2)C1
InChIKey: HDSRMYGFRHGZPX-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 > 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 508.58, ALogP 3.56, HBA 7, HBD 1, PSA 106.73, QED 0.56
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCc1cc(Br)cc(Br)c1
InChIKey: YETQQFMHUDZVIA-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of recombinant human N-terminal GST-fused KDR (D807 to V1356 residues) expressed in baculovirus infected Escherichia coli or insect Sf9 cells using poly-EY as substrate after 240 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 584.1, ALogP 7.79, HBA 5, HBD 1, PSA 56.27, QED 0.25
Compound (SMILES): C=CC(=O)Nc1ccccc1Nc1cc(N(C)C(=O)Nc2c(Cl)c(OC)cc(OC)c2Cl)ncn1
InChIKey: SNAPABPRVHZAGB-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Kinase HotSpotâ¿¢ Assay: Compounds were profiled for FGFR inhibition activity at Reaction Biology Corporation (Malvern, Pa.) with their Kinase HotSpot assay. See, Anastassiadis et al., 2011, Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29, 1039-1045.Recombinant FGFR1 (2.5 nM), FGFR2 (1 nM), FGFR3 (5 nM), or FGFR4 (12 nM) (Invitrogen) was prepared as a mixture with substrate KKKSPGEYVNIEFG (SEQ ID NO:1) (20 uM, FGFR1 substrate); and Poly [E,Y]4:1 (0.2 mg/ml, FGFR2,3,4 substrate)] in kinase reaction buffer (20 mM HEPES-HCl, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 1 mM EGTA, 0.02% Brij35, 0.1 mM Na3VO4, 0.02 mg/ml BSA, 2 mM DTT, and 1% DMSO). Compound was added to the enzyme/substrate mixture using acoustic technology (Labcyte Echo 550, Sunnyvale, Calif.) (see, Olechno et al., 2006, Improving IC50 results with acoustic droplet ejection. JALA 11, 240-246) and pre-incubated for 0, 15, or 60 minutes at room temperature. After compound pre-incubation, a mixture of ATP (Sigma-Aldrich) and 33P-γ-ATP (PerkinElmer) was added to a final concentration of 10 uM to initiate kinase reactions. Reactions were incubated for 120 minutes at room temperature and then spotted onto Whatman P81 ion exchange filter paper. Unbound phosphate was removed by extensively washing filters in 0.75% phosphoric acid. See, Anastassiadis et al., 2011, Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29, 1039-1045.
Measurement: IC50 = 1980 nM (pChEMBL 5.699999809265137)
Descriptors: MW 517.37, ALogP 5.34, HBA 7, HBD 3, PSA 117.71, QED 0.35
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(Cc3cc(F)cc(F)c3)CC2)C1
InChIKey: SWQZXCPMESABJL-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 488.55, ALogP 3.69, HBA 7, HBD 1, PSA 89.66, QED 0.45
Compound (SMILES): CCOc1ccc(CC2CC2C(=O)NO)cc1-c1nc2c(CC)nn(C)c2c(=O)[nH]1
InChIKey: HDBVIGOTKGSSOC-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 = 1920 nM (pChEMBL 5.71999979019165)
Descriptors: MW 411.46, ALogP 1.97, HBA 7, HBD 3, PSA 122.13, QED 0.4
Compound (SMILES): N#CC1CC12CCC(c1nnn3cnc4[nH]ccc4c13)CC2
InChIKey: NLCXLKGYQDXCMB-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 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 292.35, ALogP 2.79, HBA 5, HBD 1, PSA 82.66, QED 0.75
Compound (SMILES): NC(=O)c1cccc([C@H]2C[C@H]3CC[C@@H](C2)N3CCN(CCCc2ccccc2)C(=O)CO)c1
InChIKey: RBEQOHMLGWWJOC-QEIWAGNOSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Cavia porcellus
UniProt: P41144
Assay: Displacement of [3H]-DPN from guinea pig kappa opioid receptor expressed in CHO-K1 cell membranes by radioligand binding assay
Measurement: Ki = 0.5012 nM (pChEMBL 9.300000190734863)
Descriptors: MW 449.6, ALogP 2.95, HBA 4, HBD 2, PSA 86.87, QED 0.58
Compound (SMILES): On1nnc2cc(CCc3nn[nH]n3)ccc21
InChIKey: ILSPBLDTHMAASO-UHFFFAOYSA-N
Target: Catenin beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P35222
Assay: Fluorescence Polarization (FP) Assay: Experiments were performed in 96-well Microfluor 2 black plates (Waltham, Mass.), and the samples were read by a Synergy 2 plate reader (Biotek, Winooski, Vt.). The fluorescence polarization (FP) was measured at room temperature with an excitation wavelength at 485 nm and an emission wavelength at 535 nm. The FP saturation experiment was performed in an assay buffer of 137 mM of NaCl, 2.7 mM of KCl, 10 mM of Na2HPO4, 2 mM of KH2PO4, 100 ug/mL of bovine gamma globulin, and 0.01% Triton-X 100. The final reaction volume is 100 uL. The competitive FP assays used 2.5 nM of Tcf4 fluorescence tracer, 10 nM of β-catenin, and different concentrations of the tested inhibitors in 100 uL of assay buffer. Each assay plate was covered black and gently mixed on an orbital shaker for 3 h to reach equilibrium before polarization values were read. For each inhibitor competition assay, the negative controls (equivalent to 0% inhibition) refer to 2.5 nM of Tcf4 fluorescence tracer and 10 nM of β-catenin, but no tested peptide inhibitor was presented. The positive controls (equivalent to 100% inhibition) refer to only 2.5 nM of Tcf4 fluorescence tracer in a final volume of 100 uL.
Measurement: Ki = 3830 nM (pChEMBL 5.420000076293945)
Descriptors: MW 231.22, ALogP -0.03, HBA 7, HBD 2, PSA 105.4, QED 0.61
Compound (SMILES): CC1(C)CC1NC[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: YSNALLPGDSFESC-JOQLWKCKSA-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 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 338.46, ALogP 3.27, HBA 5, HBD 2, PSA 70.9, QED 0.77
Compound (SMILES): C=CC(=O)N1CCc2c([nH]c3c(C(N)=O)c(-c4ccc(Oc5ccccc5)cc4)nn23)C1
InChIKey: HCRQKHQHICVPMB-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of EGFR (unknown origin) preincubated for 1 hr followed by Biotin-AVLESEEELYSSARQ-NH2 substrate addition in presence of ATP and measured after 1 hr by TR-FRET assay
Measurement: IC50 < 0.25 nM (pChEMBL 9.60200023651123)
Descriptors: MW 427.46, ALogP 3.29, HBA 5, HBD 2, PSA 105.72, QED 0.48
Compound (SMILES): CSc1ccc(C(O)(CN[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)C(F)(F)F)cc1
InChIKey: UXHYCSPSXIPCCF-LOXQESOXSA-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 = 390 nM (pChEMBL 6.409999847412109)
Descriptors: MW 490.56, ALogP 4.39, HBA 7, HBD 3, PSA 91.13, QED 0.35
Compound (SMILES): Cc1cnc(-c2ccnc(C(C)(C)C)n2)cc1-n1c(C)cc(OCc2ccc(F)cc2F)c(Cl)c1=O
InChIKey: RAVOJDKMDLUTLW-UHFFFAOYSA-N
Target: MAP kinase-activated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P49137
Assay: Inhibition Assay: Kinase reactions is carried out in a 384-well plate (Matrical, MP101-1-PP) in 20 mM HEPES pH 7.5, 10 mM MgCl2, 0.0005% Tween-20, 0.01% BSA, 1 mM DTT, and 2% DMSO. The inhibitor concentration is varied between 0.02-30,000 nM, while the Hsp27 peptide substrate and MgATP are held constant at 1 μM and 10 μM, respectively. Activated p38α is added to a final concentration of 20 pM for reactions with nonphosphorylated 1 nM His6-MK2 in the cascade reaction. For the p38α/PRAK cascade, unactivated GST-PRAK is held constant at 1 nM while p38α is added in to a final concentration of 20 pM. Kinase reactions are incubated at room temperature and quenched after 30 minutes by the addition of stop buffer (180 mM HEPES, 30 mM EDTA, and 0.2% Coating Reagent-3). Under these conditions, approximately 20% of the substrate Hsp27 peptide is phosphorylated. Reactions are initiated by the addition of activated p38α except for preincubation experiments, where reactions are initiated by the addition of Hsp27 peptide and MgATP. Preincubation of p38α with inhibitor or p38α with unactivated His6-MK2 or unactivated GST-PRAK and inhibitor are performed at 2X final assay concentrations at room temperature 240 minutes prior to adding ATP and Hsp27 peptide to initiate catalysis.
Measurement: IC50 = 210 nM (pChEMBL 6.679999828338623)
Descriptors: MW 510.97, ALogP 6.11, HBA 6, HBD 0, PSA 69.9, QED 0.32
Compound (SMILES): CCCCCC(O)c1ccc(N2C(=O)CC[C@@H]2C=CCc2ccc(C(=O)O)s2)cc1
InChIKey: WWZDFNDWWMQENR-YMXDCFFPSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: Displacement of [3H]-PGE2 from human EP4 receptor expressed in HEK293 cells after 60 mins
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 427.57, ALogP 5.35, HBA 4, HBD 2, PSA 77.84, QED 0.4
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCCC1CNC(C)(C)CO1
InChIKey: DCHUHENWAKOFLF-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused ErbB2 kinase domain (Q679 to V1255 residues) expressed in baculovirus infected insect Sf9 cells using biotinylated poly-EY as substrate after 60 mins by alphascreen assay
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 477.39, ALogP 5.22, HBA 7, HBD 2, PSA 77.53, QED 0.48
Compound (SMILES): OCCOc1ccc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: ZTJGFWUKYIOSTN-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 392.46, ALogP 2.36, HBA 7, HBD 2, PSA 91.57, QED 0.52
Compound (SMILES): COc1cc(F)ccc1-c1nc(Nc2cc(F)cc(CS(C)(=N)=O)c2)ncc1F
InChIKey: NSJOYQIOHVHMCZ-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P24941
Assay: CDK2/CycE Kinase Assay: Recombinant fusion proteins of GST and human CDK2 and of GST and human CycE, expressed in insect cells (Sf9) and purified by Glutathion-Sepharose affinity chromatography, were purchased from ProQinase GmbH (Freiburg, Germany). 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 CDK2/CycE 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 = 220 nM (pChEMBL 6.659999847412109)
Descriptors: MW 422.43, ALogP 4.49, HBA 6, HBD 2, PSA 87.96, QED 0.61
Compound (SMILES): O=C(O)[C@H]1[C@@H]2C[C@@H](C(=O)N3CC[C@](c4ccc(C(F)(C(F)(F)F)C(F)(F)F)cc4)(S(=O)(=O)c4ccc(F)cc4)C3)C[C@@H]21
InChIKey: HNOZQRKROBDUDI-LPSQLEIASA-N
Target: Nuclear receptor ROR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35398
Assay: Inverse agonist activity at human Gal4-fused RORalpha LBD (272 to 556 residues) expressed in human Jurkat cells measured after 18 hrs by steady-glo luciferase reporter gene assay
Measurement: EC50 > 40000 nM (pChEMBL 4.3979997634887695)
Descriptors: MW 625.53, ALogP 5.37, HBA 4, HBD 1, PSA 91.75, QED 0.34
Compound (SMILES): Cc1cc(Nc2nc([C@H](O)c3ccc(F)cc3)nc3ccccc23)n[nH]1
InChIKey: DCRWIATZWHLIPN-QGZVFWFLSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Competition Binding Assay: For the binding assays, streptavidin-coated magnetic beads were treated with biotinylated affinity ligands for 30 min at room temperature to generate affinity resins. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinase, liganded affinity beads, and test compounds in 1x binding buffer (20% SeaBlock, 0.17xPBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100x stocks in DMSO and rapidly diluted into the aqueous environment. DMSO was added to control assays lacking a test compound. Primary screen interactions were performed in polypropylene 384-well plates in a final volume of 34 uL, while Kd determinations were performed in polystyrene 96-well plates in a final volume of 135 uL. The assay plates were incubated at room temperature with shaking for 1 hr, long enough for binding reactions to reach equilibrium, and the affinity beads were washed extensively with wash buffer (1xPBS, 0.05% Tween 20) to remove unbound protein. The beads were then resuspended in elution buffer (1xPBS, 0.05% Tween 20, 2 uM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 min. The kinase concentration in the eluates was measured by quantitative PCR. Each kinase was tested individually against each compound.
Measurement: Kd = 8800 nM (pChEMBL 5.059999942779541)
Descriptors: MW 349.37, ALogP 3.63, HBA 5, HBD 3, PSA 86.72, QED 0.52