lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ncc(CN2CCC(c3ccc4[nH]c(-c5cc(C(C)(C)O)c6ncnn6c5)c(C(C)C)c4c3)CC2)cn1
InChIKey: RYUASKZIYSNIID-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 9234 nM (pChEMBL 5.039999961853027)
Descriptors: MW 523.69, ALogP 5.71, HBA 7, HBD 2, PSA 95.23, QED 0.3
Compound (SMILES): CCc1cnc(-c2cc(Cl)ccc2F)cc1Nc1ccncc1C(=O)NC[C@H](C)O
InChIKey: OGPVQKHTKOAKDQ-ZDUSSCGKSA-N
Target: Bone morphogenetic protein receptor type-1A (SINGLE PROTEIN), Homo sapiens
UniProt: P36894
Assay: In Vitro Kinase Assay Inhibition of ALK1/2/3/4/5/6 Kinases: Compound of the invention were screened in an in vitro kinase assay against several members of the TGFβ family of Ser/Thr kinases. The kinases tested were ALK1 (ACVRL1), ALK2 (ACVR1), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFBR1), and ALK6 (BMPR1B). Standard kinase testing conditions and techniques were employed. For each case, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer. Compound of the invention were delivered into the reaction, followed 15-20 min later by addition of a mixture of ATP and 33P ATP to a final concentration of 10 μM. Reactions were carried out at RT for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper. Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. Kinase activity data was expressed as the percent of remaining kinase activity in test samples compared to vehicle. IC50 values were generated from activity values performed at multiple concentrations.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 428.9, ALogP 4.35, HBA 5, HBD 3, PSA 87.14, QED 0.52
Compound (SMILES): Cc1cnc(-c2cc(OC3COC3)cc(C(=O)N[C@H](C)c3nnc(C(F)(F)F)s3)c2)s1
InChIKey: CAKLWYTTWVXMCY-SNVBAGLBSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Biological Assay: A fluorescent imaging plate reader (FILEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Rim-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C.
Measurement: IC50 = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 470.5, ALogP 4.26, HBA 8, HBD 1, PSA 86.23, QED 0.58
Compound (SMILES): COc1ccc(-c2cncc(C3COB(O)C3)c2)cc1OC1CC1
InChIKey: SIWUJTCBTPZDQD-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Cytokine Assays: Cytokine inhibitory activity is determined by measuring the effect of test agent on the release of the cytokines IL-4, IL-13 and IFNγ from human peripheral blood mononuclear cells (PBMCs) stimulated with the T-cell mitogen phytohemagglutinin-L (PHA-L). At the time of assay, human PBMCs (Astarte Biologics) are removed from cryopreservation, thawed quickly at 37° C., diluted in assay medium (RPMI-Gibco 1640 medium, 10% heat inactivated fetal bovine serum (HIFBS), 2 mM glutamine and 10 mM HEPES pH 7.4) and then centrifuged at 250×g for 5 minutes. The resulting cell pellet is re-suspended in assay medium to a concentration of approximately 5×106 cells/mL and 50 μL of this cell suspension (approximately 250,000 cells) is added to each well of a 384-well cell culture microtiter plate (Perkin Elmer). Varying concentrations of test compound are then diluted in assay medium and added to the assay plate wells in a volume of 25 μL. After −10 minutes at room temperature (21° C.) the cells are stimulated by the addition of 25 μL of PHA-L (14 μg/mL; Millipore) and the assay plate is placed in an incubator at 37° C. in a humidified environment in 5% carbon dioxide. The final assay conditions are approximately 250,000 human PBMCs per well in assay medium containing 3.5 μg/mL PHA-L and the indicated final concentration of test compound (approximately 10 μM to 9.5 μM by 4-fold dilution). The final concentration of DMSO in the assay is approximately 0.25%. After 48 hours, the assay plate is removed from the incubator and centrifuged at 250×g for 5 minutes. A portion of the resulting cell supernatant is then used to determine the amount of IL-4, IL-13 and IFNγ in each well. Cytokine measurements are made using human IL-4, IL-13 or IFNγ HTRF assay kits (CisBio) following the manufacturer's assay protocol. The concentrations and resulting effect values for tested compounds are plotted and the concentration of compound required for 50% effect (IC50) is determined with a four-parameter logistic dose response equation (E-WorkBook, ID Business Solutions Ltd.).
Measurement: IC50 = 0.63 nM (pChEMBL 9.199999809265137)
Descriptors: MW 325.17
Compound (SMILES): C=CC(=O)Nc1cccc(-c2cccc3cnc(Nc4ccc(NC5CN(CCO)C5)cc4)nc23)c1
InChIKey: ZZSYZBGNOCCFDT-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In vitro assay: The compound of Formula I binds to a kinase selected from the group consisting of EGFR, EGFR L858R, EGFR T790M, EGFR del E746-A750, or EGFR L858R/T790M mutant, Her2, Her4, Fak, FGFR1, FGFR2, FGFR3, FGFR4, Btk, Met, Pim1, Pim2, Pim3, Pyk2, KDR, Src and Ret, and any mutated versions thereof with a Kd which is lower than 50 μM, 25 μM, 10 μM, 5 μM, or 1 μM as measured in an in vitro assay. In some embodiments, the compound of Formula I binds to a kinase selected from the group consisting of Btk, KDR, EGFR, EGFR L858R, EGFR T790M or EGFR L858R/T790M mutant with a Kd which is lower than 50 μM, 25 μM, 10 μM, 5 μM, or 1 μM as measured in an in vitro assay. For example, the compound of Formula I binds to a kinase which is EGFR, EGFR L858R, EGFR T790M, EGFR del E746-A750, EGFR L858R/T790M mutant with a Kd which is lower than 50 μM, 25 μM, 10 μM, 5 μM, or 1 μM as measured in an in vitro assay.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 480.57, ALogP 4.25, HBA 7, HBD 4, PSA 102.41, QED 0.27
Compound (SMILES): C=CC(=O)N1CCN(CCn2c(=O)ccc3cnc(Nc4cnn(CCF)c4)nc32)[C@@H](C)C1
InChIKey: ORYCIGRTFGOJIW-INIZCTEOSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Biochemical JAK and Tyk2 Kinase Assays: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies. Serially or discretely diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for 1 h. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for 1 h before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.
Measurement: Ki = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 454.51, ALogP 1.42, HBA 9, HBD 1, PSA 101.18, QED 0.51
Compound (SMILES): O=C(O)CC1CCC(c2ccc(Nc3nc(N4CCCCCC4)nc4cn[nH]c(=O)c34)cc2)CC1
InChIKey: OILJTSQUFHFIEV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzymatic JAK2 Kinase Activity: Two μL of 10% DMSO in blank, substrate control and positive control wells and 2 μL of test compound in test wells was added. Thirteen μL of assay buffer in blank and substrate control wells and 13 μL of Enzyme buffer mix in positive and test wells was added. The reaction mixture was incubated for 30 minutes at RT on a plate shaker. Ultra Light-pGT substrate (5 μL) [poly Glu-Tyr (4:1) labeled with U Light dye, a tyrosine kinase substrate] and ATP mix was added to all wells. The reaction plate was incubated for 60 minutes at RT on a plate shaker. The reaction was stopped by adding 40 mM EDTA (10 μL) in buffer. Ten μL of antibody was added to all the wells. The plate was read in a Wallac® 1420 Multilabel Counter Victor 3 instrument (Ex: 340 nm Em: 615 & 665 nm)
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 476.58, ALogP 4.59, HBA 7, HBD 3, PSA 124.1, QED 0.47
Compound (SMILES): COc1cccnc1CN1CCN(c2nc3c(c(=O)[nH]2)CCCN3C)CC1
InChIKey: SSDOGFZFEYCRRH-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Enzyme Assay: The Poly ADP-ribose polymerase 1(PARP1) assay is an ELISA which detects poly ADP ribose incorporated into plate-bound histone protein in a 384-well format. Using recombinant hPARP1 enzyme, this assay uses biotinylated NAD+ and measures the incorporation into histone using a streptavidin-horseradish peroxidase (HRP) conjugate and TMB peroxidase substrate to generate a colorimetric signal.Histone is diluted to 0.1 mg/ml in coating buffer (50 mM Na2CO3, pH 9.4, Mallinckrodt, St. Louis, Mo.) and 25 μl is added to each well of a Corning 3700 plate. The plates are incubated overnight at 4° C. The next day the plates are washed 3× with 50 μl/well wash buffer (PBS (prepared from 10× concentrate) with 0.1% Tween-20) followed with blocking for 1.5 hours at room temperature using 50 μl 1% Casein block buffer in 1×PBS (Roche, Indianapolis, Ind. #11666789001). After blocking, the plates are washed 3 times with 50 μl/well wash buffer. The PARP1 enzyme assay is set up by using 0.01 U/μl hPARP1 (Trevigen, Gaithersburg, Md. #4668-500-01), 0.5×PARP cocktail, activated DNA (Trevigen, Gaithersburg, Md. #4671-096-03 and #4671-096-06) and compounds diluted from 10 μM to 4 nM final concentration in Assay buffer containing 50 mM Tris, pH 8.0, 10 mM MgCl2, 1 mM DTT (Invitrogen, Grand Island, N.Y. #15508-013), 0.5% Triton X-100 and 1.0% DMSO. The reaction is incubated for 60 minutes at room temperature and stopped by washing the plate 3 times with 50 μl/well wash buffer. Detection of the biotin-NAD+ incorporation is done using 25 μl/well of Strepavidin-HRP diluted 1:3000 with wash buffer and incubated for 60 minutes at room temperature.
Measurement: IC50 = 15300 nM (pChEMBL 4.820000171661377)
Descriptors: MW 370.46, ALogP 0.88, HBA 7, HBD 1, PSA 77.59, QED 0.86
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc(N)c(/C=C/c4ccccc4)c3N(C)C2=O)c1F
InChIKey: YKBROVXUBANFCS-MDZDMXLPSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).FGFR1 and FGFR2 were measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 μM, respectively and FGFR2, 0.01 nM and 100 μM, respectively. The enzymes were purchased from Millipore or Invitrogen.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 452.46, ALogP 4.71, HBA 5, HBD 1, PSA 80.92, QED 0.61
Compound (SMILES): Clc1ccc(OC2CN(c3ccccc3)C2)c(OC2CNC2)c1
InChIKey: ZKYIYIJTVODBMI-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2C (SINGLE PROTEIN), Homo sapiens
UniProt: P28335
Assay: Binding Assay: Receptor binding was performed using membrane fractions prepared from the HEK-293 cell line recombinantly expressing rat 5-HT7 receptors (NCBI accession NM_022938). Compound affinity for the rat 5-HT7 receptor subtype was evaluated by competitive radioligand binding assays using 5-carboxamido[3H]tryptamine ([3H]5-CT) (Amersham Biosciences, cat. 90000403) detection. HitHunter cAMP assays are in-vitro based competitive immunoassays. The assay was performed on the HEK-293 cell line stably transfected with r5-HT7 receptor. Cells were pre-incubated with test compounds for 10 minutes. For antagonist testing, the cells were then challenged with 100 nM 5-CT for 20 minutes. Cells were then lysed and cAMP measured according to manufacturers protocol (Amersham, cat. NET791250UC) or [3H]mesulergine (Amersham, cat. TRK1041). The assay was performed on membranes prepared from HEK-293 cells stably transfected with h5-HT6. Following centrifugation, membranes were resuspended and incubated for 60 min at room temperature with 1.7 nM [3H]LSD in the presence of increasing concentration of test compounds. Nonspecific binding was defined in the presence of 10 μM clozapine (Tocris, cat. TRK1068). Homogenized HEK-293 membranes expressing the human SERT were incubated in 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 5 mM KCl with [3H]-citalopram (3 nM) with or without test compounds. Nonspecific binding was determined in the presence of 10 μM fluoxetine. Radioactivity readouts and Ki values were performed as previously described for r5-HT7.
Measurement: Ki = 1009 nM (pChEMBL 6)
Descriptors: MW 330.82, ALogP 2.96, HBA 4, HBD 1, PSA 33.73, QED 0.91
Compound (SMILES): O=C(NCC1(O)CCCC1)[C@H]1CCN(c2ncnc3[nH]ccc23)CC12CC2
InChIKey: IOIPOBMOUFWACW-OAHLLOKOSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Kinase Assays: Human baculovirus-expressed JAK1, 2, 3 and TYK2 were purchased from Carna Biosciences, Inc. All four purified enzymes contain only the catalytic domain. JAK1 (aa 850-1154) and TYK2 (aa 871-1187) are expressed with an N-terminally fused GST-tag, and JAK2 and JAK3 with an N-terminally fused His-tag.Inhibition of phosphorylation of a synthetic peptide was measured in an HTRF-based assay using the TK substrate-Biotin from the Cisbio HTRFKinEASE TK kit. First, 2 μl of TK solution (TK substrate-biotin in kinase buffer [1× enzymatic buffer from HTRFKinEASE TK kit, 1 mM DTT]) is added to a plate containing 1 μl prediluted compound (final assay concentration DMSO: 0.75%). Then, 5 μl kinase-ATP mix (prepared in kinase buffer) is added to the wells and the plates are incubated at RT for 20-30 min. For all four kinases a concentration of ATP that corresponded to the Km for ATP was used. The final concentrations of buffers, substrate, kinase and ATP were: JAK1: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 10 mM MgCl2, 1 mM DTT, 7 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 5 ng JAK1; JAK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 4 μM ATP, 1 μM TK Substrate-Biotin and 0.1 ng JAK2; JAK3: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 2 μM ATP, 1 μM TK Substrate-Biotin and 0.3 ng JAK3; TYK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 13 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 0.8 ng TYK2. Thereafter, the kinase reaction is stopped by adding 4 μl detection mix (final concentrations: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 0.8 M KF, 20 mM EDTA, 42 nM Streptavidin-XL665 and 1:400 STK Ab Cryptate) and the plates are incubated overnight in the dark. The HTRF signal is read using an Envision plate reader.
Measurement: EC50 = 26.1 nM (pChEMBL 7.579999923706055)
Descriptors: MW 369.47, ALogP 1.99, HBA 5, HBD 3, PSA 94.14, QED 0.77
Compound (SMILES): C[C@@H]1C[C@H](N)C[C@H](c2ccncc2NC(=O)c2ccc(F)c3cc(N4C[C@@H](O)C[C@@H]4C)cnc23)C1
InChIKey: GHVQZKXLUQXDJN-JNIAIEOXSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA,) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was preincubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 477.58, ALogP 4.21, HBA 6, HBD 3, PSA 104.37, QED 0.52
Compound (SMILES): COCCOC(=O)N1[C@@H]2CCC[C@H]1C[C@H](Nc1nc(Nc3cc(C)[nH]n3)cc3ncccc13)C2
InChIKey: ORORFPPZLZDASP-NNZMDNLPSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Biochemical JAK Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for lh. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for lh before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.Test compounds having a higher pKi value in each of the four JAK assays show greater inhibition of JAK activity. Compounds of the invention tested in this assay typically exhibited pKi values between about 7 and about 10.3
Measurement: Ki = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 465.56, ALogP 3.99, HBA 8, HBD 3, PSA 117.29, QED 0.45
Compound (SMILES): CS(=O)(=O)c1cccc(N2CCN(Cc3ccc(C(F)(F)F)c(Cl)c3)[C@@H](c3ccccc3)C2)c1
InChIKey: SYWHAWSOSMSFBS-XMMPIXPASA-N
Target: Oxysterols receptor LXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q13133
Assay: Human LXRalpha and LXRbeta Coactivator Recruitment FRET Assay: This assay is based on the ability of the LXR-LBDs (LXRα and LXRβ) to recruit and interact with a co-activator peptide. This assay was run in agonist mode, to characterize compounds of the invention that exhibit LXR agonist activity, and in antagonist mode to characterize compounds of the invention having LXR antagonist activity and which cause a concentration-dependent release of the co-activator peptide in the presence of the full LXR agonist T0901317. The interaction between glutathione S-transferase (GST)-tagged, recombinant human LXRα-LBD or LXRβ-LBD and fluorescein-conjugated co-activator peptide SRC2-3 (Cat# PV4588, Life Technologies, Grand Island, N.Y.) was measured via time resolved-fluorescence resonance energy transfer (TR-FRET). The assay was performed in 96-well half-area black Opti-plates (Cat# 3686, Corning, Lowell, Mass.) in 20 mM TRIS-HCl buffer, pH 8, containing 150 mM NaCl and 5 mM DDT in a total volume of 80 μL. Test compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions were also prepared in DMSO, as 40× solutions.
Measurement: EC50 = 479 nM (pChEMBL 6.320000171661377)
Descriptors: MW 508.99, ALogP 5.83, HBA 4, HBD 0, PSA 40.62, QED 0.43
Compound (SMILES): CC(C)(O)Cn1cc(-c2nc(-c3cnn4ccccc34)nc(N[C@@H]3CCCN(C(=O)CO)C3)c2F)cn1
InChIKey: PPPZKRNWVZENFR-QGZVFWFLSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK kinase Assays: The catalytic domains of human JAK1 (aa 850-1154), JAK2 (aa 826-1132), JAK3 (aa 795-1124) and Tyk2 (aa 871-1187) were expressed as N-terminal GST-fusion proteins using a baculovirus expression system and were purchased from Carna Biosciences. The enzymatic activity was assayed using as substrate a biotinylated peptide, poly (GT)-Biotin (CisBio). The peptide concentration in the reactions was 60 nM for JAK1, 20 nM for JAK2, 140 nM for JAK3 and 50 nM for Tyk2. The degree of phosphorylation was detected by TR-FRET (time-resolved fluorescence energy transfer).IC50s of compounds were measured for each kinase in a reaction mixture containing the enzyme, ATP and the peptide in 8 mM MOPS (pH 7.0), 10 mM MgCl2, 0.05% β-mercaptoethanol, 0.45 mg/ml BSA. The ATP concentration in the reactions was 3 μM for JAK1, 0.2 μM for JAK2, 0.6 μM for JAK3 and 1.8 μM for Tyk2. The enzymatic reactions took place for 30 minutes at room temperature. Then, the reactions were stopped with 20 μL of quench detection buffer (50 mM HEPES, 0.5 M KF, EDTA 0.25 M, 0.1% (w/v) BSA, pH 7.5) containing 0.115 μg/mL of anti-phosphoTyr (PT66)-Cryptate (CisBio) and a variable concentration of SA-XL665 (CisBio) to keep the SA-B ratio constant. Incubate for 3 h and read on Victor 2V spectrofluorometer (Perkin Elmer) set to read fluorescence resonance energy transfer.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 508.56, ALogP 1.96, HBA 10, HBD 3, PSA 133.7, QED 0.35
Compound (SMILES): COc1ccc(-c2ccc3nccc(C(=O)NCC(=O)N4CC(F)(F)C[C@H]4C#N)c3c2)cc1
InChIKey: GLDJFLAXVMANDQ-KRWDZBQOSA-N
Target: Dipeptidyl peptidase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P27487
Assay: DPPIV Enzymatic Activity Assay: DPPIV enzymatic activity assay. To assay baseline dipeptidyl peptidase-4 (DPPIV) activity, 40 ng of recombinant human DPPIV (rhDPPIV) (R&S system, #1180-SE) or 40 ng of recombinant mouse DPPIV (rmDPPIV) (R&S system, #954-SE) was incubated with 400 μM of H-Gly-Pro-pNA substrate (BACHEM, #L-1880) in a DPPIV assay buffer (25 mM Tris, pH 8.3) for 30 min at 37° C. protected from the light in 96-well black plates (Nunc, #237108). To assay DPPIV inhibition by test compounds, test compounds were pre-incubated with the enzyme for 15 min at 37° C. before starting the reaction by substrate addition in 96-well black plates (Nunc, #237108). Para-nitroaniline (pNA) release was detected by measuring absorbance at 405 nm using a Multifunction Microplate Reader (Synergy 4, Biotek). All measurements were carried out in triplicate. Val-boroPro, a non-specific prolyl peptidase inhibitor, was used as a positive control.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 450.45, ALogP 3.4, HBA 5, HBD 1, PSA 95.32, QED 0.64
Compound (SMILES): Cc1cn2c([C@@H]3CCCOC3)ncc2c(=O)n1Cc1cccc(F)c1
InChIKey: PZKOOBRKCGIRJF-OAHLLOKOSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1C (SINGLE PROTEIN), Homo sapiens
UniProt: Q14123
Assay: PDE1 Inhibition Assay: PDE1A, PDE1B and PDE1C assays were performed as follows: the assays was performed in 60 μL samples containing a fixed amount of the PDE1 enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES pH 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 15 nM tritium labelled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 h at room temperature before being terminated through mixing with 20 μL (0.2 mg) yttrium silicate SPA beads (PerkinElmer). The beads were allowed to settle for 1 h in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC50 values were calculated using XIFit (model 205, IDBS).
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 341.39, ALogP 2.89, HBA 5, HBD 0, PSA 48.53, QED 0.74
Compound (SMILES): CCc1c(Cc2ccccc2)cnc2c1CC[C@@H]1CNCCN21
InChIKey: NZMUSZUGSVODII-QGZVFWFLSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Homo sapiens
UniProt: P28223
Assay: Radioligand Binding Assay: Radioligand binding assays were performed using the commercially available 5-HT2 receptor agonist [125I]DOI as the radioligand and nonspecific binding was determined in the presence of unlabeled DOI at a saturating concentration of 10 μM. Competition experiments utilized 5-HT2 receptor expressing HEK293 cell membranes obtained as described in Example 3 (15-25 μg membrane protein/well) and radioligand at final assay concentrations of 0.4 to 0.6 nM. Experiments comprised addition of 95 μL of assay buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2), 50 μL of membranes, 50 μL of radioligand stock, and 5 μL of test compound diluted in assay buffer to 96-well microtiter plates, which were then incubated for 1 h at room temperature. Assay incubations were terminated by rapid filtration through PerkinElmer F/C filtration plates under reduced pressure using a 96-well Packard filtration apparatus, followed by washing three times with ice cold assay buffer. Plates were then dried at 45° C. for a minimum of 2 h. Finally, 25 μL of BetaScint scintillation cocktail was added to each well and the plates were counted in a Packard TopCount scintillation counter. In each competition study, test compounds were dosed at ten concentrations with triplicate determinations at each test concentration.
Measurement: Ki = 36.7 nM (pChEMBL 7.429999828338623)
Descriptors: MW 307.44, ALogP 2.96, HBA 3, HBD 1, PSA 28.16, QED 0.94
Compound (SMILES): C[C@]1(O)CC[C@H](CNc2ccc(S(=O)(=O)NC(=O)c3ccc(N4CCC5(CC[C@@H](N6CCC[C@H]6c6ccccc6C6CC6)C5)C4)cc3Oc3cnc4[nH]ccc4c3)cc2[N+](=O)[O-])CC1
InChIKey: DPGZYKGGBPOPMZ-PFOUOBOSSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Bcl-2/Bcl-X TR-FRET Assay: Compounds disclosed herein were tested for blocking of Bcl-2/Bcl-X protein with its ligand in an assay based on Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) methodology. Recombinant human 0.05 nM Bcl-2/0.03 nM Bcl-X protein was pre-incubated with a serial dilution of compounds disclosed herein (maximum concentration is 0.1 μM for Bcl-2 assay, and 10 μM for Bcl-xl assay, 3-fold serially diluted, 10 points; or maximum concentration is 0.02 μM for Bcl-2 assay, and 2 μM for Bcl-xl assay, 3-fold serially diluted, 10 points) at room temperature for 0.5 hour in an assay buffer containing 20 mM potassium phosphate buffer, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.05% Tween-20, 0.01% BSA. Then the FITC labeled Bak peptide Ac-GQVGRQLAIIGDK(FITC)INR-amide (0.5 nM for Bcl-2, 0.3 nM for Bcl-xl) and MAb Anti 6His Tb cryptate Gold were added to plate and further incubated at room temperature for 1 hour. The TR-FRET signals (337 nm-520 nm-490 nm) were read on BMG PHERAstar FSX instrument. The inhibition percentage of Bcl-2/Bcl-X interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the TR-FRET signals. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software. To improve the assay sensitivity and test more potent compounds in the present application, the bcl-2 concentration was reduced in method B.
Measurement: IC50 = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 888.1, ALogP 9.2, HBA 11, HBD 4, PSA 183.03, QED 0.07
Compound (SMILES): COc1cc2ncnc(Sc3cccc(NC(=O)Nc4cc(C(C)(CF)CF)nn4-c4ccccc4)c3)c2cc1OC
InChIKey: ROTKIWQMMHTKON-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Competition Binding Assay: Competition binding assays used herein were developed, validated and performed as described in Fabian et al., Nature Biotechnology 2005, 23, 329-336. Kinases were produced as fusions to T7 phage (See, Fabian et al. or WO04/015142) or alternatively, the kinases were expressed in HEK-293 cells and subsequently tagged with DNA for PCR detection (See, WO08/005310). 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 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100× 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 μL, while Kd determinations were performed in polystyrene 96-well plates in a final volume of 135 μL. The assay plates were incubated at room temperature with shaking for 1 hour, long enough for binding reactions to reach equilibrium, and the affinity beads were washed extensively with wash buffer (1×PBS, 0.05% Tween 20) to remove unbound protein. The beads were then resuspended in elution buffer (1×PBS, 0.05% Tween 20, 2 μM 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. KdS were determined using eleven serial threefold dilutions.
Measurement: Kd > 1000 nM (pChEMBL 6)
Descriptors: MW 590.66, ALogP 6.82, HBA 8, HBD 2, PSA 103.19, QED 0.17
Compound (SMILES): COc1ccc(CN(C)C(=O)NC2CCC(CS(=O)(=O)NNC(=O)c3cc(-c4cn(C)cn4)cc(Cl)c3F)C2)cc1
InChIKey: CIKBFTZNNWUQDF-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT7 (SINGLE PROTEIN), Homo sapiens
UniProt: O95251
Assay: KAT-7 Enzyme Assay: Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-7 enzyme (6 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 2 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 120 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 607.11, ALogP 3.46, HBA 7, HBD 3, PSA 134.66, QED 0.3
Compound (SMILES): C[C@@H]1Cc2c([nH]c3ccccc23)[C@@H](c2c(F)ccc(OCCNCCCF)c2C(F)F)N1C[C@H](C)C(=O)O
InChIKey: SHBYLUMKVZHSKT-PAPQREIWSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: hERG Binding Assay: hERG (human ether a go go-related gene) potassium channels are essential for normal electrical activity in the heart. Arrhythmia can be induced by a blockage of hERG channels by a diverse group of drugs. This side effect is a common reason for drug failure in preclinical safety trials [Sanguinetti et al., Nature, 2006, 440, 463-469.] and therefore minimisation of hERG channel blocking activity may be a desirable property for drug candidates.The purpose of the hERG binding assay is to evaluate the effects of test compounds on the voltage-dependent potassium channel encoded by the human ether go go-related gene (hERG) using a constitutively expressing CHO cell line on the Nanion Syncropatch 384PE automated patch clamp system.
Measurement: IC50 > 40000 nM (pChEMBL 4.3979997634887695)
Descriptors: MW 535.58, ALogP 5.63, HBA 4, HBD 3, PSA 77.59, QED 0.21
Compound (SMILES): C[C@H](NC(=O)c1c(N)nn2cccnc12)c1cc2cccc(Cl)c2c(=O)n1CCc1nnnn1C
InChIKey: LDVDIZLJSHEFSX-LBPRGKRZSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition Assay: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110δ/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/ml). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration of 10 or 100 μM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 μL 1N HCl followed by 160 μL CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid. The TLC plates are then dried, exposed to a phosphorimager screen (Storm, Amersham), and quantitated. For each compound, kinase activity is measured at 10-12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (typically, 200 μM). For compounds showing significant activity, IC50 determinations are repeated two to four times, and the reported value is the average of these independent measurements.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 492.93, ALogP 1.54, HBA 11, HBD 2, PSA 150.91, QED 0.36
Compound (SMILES): OC(c1cc(-c2ncnc3cc(N4CCOCC4)ccc23)ccc1Cl)c1ncncc1F
InChIKey: LGZAMQCFFYGHBM-UHFFFAOYSA-N
Target: DNA-dependent protein kinase catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P78527
Assay: Biochemical Assay: The kinase assay was carried out in streptavidin-coated 348-well microtitre flashplates. To this end, 1.5 ug of DNA-PK/protein complex and 100 ng of biotinylated substrate, such as, for example, PESQEAFADLWKK-biotin-NH2 (biotin-DNA-PK peptide), were incubated for 90 min at room temperature in a total volume of 36.5 ul (34.25 mM HEPES/KOH; 7.85 mM Tris HCl; 68.5 mM KCl; 5 uM ATP; 6.85 mM MgCl2; 0.5 mM EDTA; 0.14 mM EGTA; 0.69 mM DTT; pH 7.4) with 500 ng of DNA from calf thymus, 0.1 uCi of 33P-ATP and 1.8% of DMSO per well with and without the test compound. The reaction was stopped using 50 ul/well of 200 mM EDTA. After incubation for a further 30 min at room temperature, the liquid was removed. Each well was washed three times with 100 ul of 0.9% saline solution. A nonspecific reaction (blank value) was determined using 10 uM of an innate kinase inhibitor. The radioactivity measurement was carried out using a TopCount. IC50 values were calculated in RS1.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 451.89, ALogP 3.8, HBA 7, HBD 1, PSA 84.26, QED 0.5
Compound (SMILES): C=CC(=O)N1CC(CCn2c(=O)ccc3cnc(Nc4ccc(N5CCN(C(C)=O)[C@H](C)C5)cc4)nc32)C1
InChIKey: RLLIUVJQKZFJBZ-LJQANCHMSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Biochemical JAK and Tyk2 Kinase Assays: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies. Serially or discretely diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for 1 h. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for 1 h before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.
Measurement: Ki < 0.1 nM (pChEMBL 10)
Descriptors: MW 515.62, ALogP 2.63, HBA 8, HBD 1, PSA 103.67, QED 0.48
Compound (SMILES): CC(=O)N1CCN(S(=O)(=O)C[C@H]2C[C@@H](N(C)c3ncnc4[nH]ccc34)C2)CC1
InChIKey: IUKJDEJWJMTDOO-GASCZTMLSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 406.51, ALogP 0.67, HBA 6, HBD 1, PSA 102.5, QED 0.78
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(CC(C)(C)O)CC4)cc3c2C(C)C)cn2ncnc12
InChIKey: ODHMTZJYIOWWCT-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 3063 nM (pChEMBL 5.510000228881836)
Descriptors: MW 445.61, ALogP 5.26, HBA 5, HBD 2, PSA 69.45, QED 0.44
Compound (SMILES): CC(C(=O)Nc1cc(C2CC2)[nH]n1)c1cccc(-c2ccc(NC(=O)/C=C/CN3CC[C@H](F)C3)nc2)c1
InChIKey: FELGDXDUEDTBOE-NEKWBUCNSA-N
Target: CDK7/Cyclin H/MNAT1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P51948
Assay: Biochemical Assay: The ability of compounds to inhibit CDK7 kinase activity was tested in a TR-FRET assay using 5 nM of CDK7/CycH/MNAT1 obtained from Invitrogen, USA. Test compounds were pre-incubated with the kinase at room temperature for 60 min. After the incubation, substrate mix [100 nM Ultra-light MBP (Perkin Elmer, USA) and 1 mM ATP (Sigma)] was added. The above reaction was stopped by the addition of 40 mM EDTA after 60 minutes of kinase reaction. 1 nM Eu-labelled antiphospho-MBP antibody [Perkin Elmer, USA] was added, mixed well and the fluorescence emission at 615 nm and 665 nm [excitation at 340 nm] was measured. The final DMSO concentration in the assay was 1%. For IC50 determination, appropriate concentrations were made by ⅓rd serial dilutions of 10 mM DMSO stock solution of test compound. All the fluorescence measurements were made in a Victor 3 Multilabel Counter [Perkin Elmer, USA]. The IC50 was determined by fitting the dose response data to sigmoidal curve fitting equation using GraphPad Prism software V5. To identify compounds that inhibit CDK7 irreversibly, time depended inhibition studies were carried by pre-incubating compound with the enzyme at three time points (20, 60 and 180 min) and carrying out assay as described above.
Measurement: IC50 = 62.5 nM (pChEMBL 7.199999809265137)
Descriptors: MW 502.59, ALogP 4.63, HBA 5, HBD 3, PSA 103.01, QED 0.37
Compound (SMILES): Cc1cc(Oc2ccccc2)ccc1N1C(=O)Nc2c(C(=O)N[C@H]3CCCOC3)sc3nccc1c23
InChIKey: FIMYKNKLIGEZMM-KRWDZBQOSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: TBD: TBD
Measurement: IC50 = 501 nM (pChEMBL 6.300000190734863)
Descriptors: MW 500.58, ALogP 5.99, HBA 6, HBD 2, PSA 92.79, QED 0.35
Compound (SMILES): Cn1cc(Nc2nccc(N3C[C@H]4CC[C@@H](C3)N4C3(CC#N)COC3)n2)cn1
InChIKey: JSOOKLFPJNXYMJ-IYBDPMFKSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Caliper Enzyme Assay: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 155 nM (pChEMBL 6.809999942779541)
Descriptors: MW 380.46, ALogP 1.29, HBA 9, HBD 1, PSA 95.13, QED 0.83