lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Nc1ccc(-c2cccs2)cc1NC(=O)C1CCCCO1
InChIKey: QUBMXRRMOBLEGI-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: Inhibition of Histone Deacetylase Enzymatic Activity Assay: HDAC2: The following trypsin-coupled protocol and Caliper protocol described Herein was used to assay the compounds of the invention. HDAC2-Kinetic IC50 3Hr PreIncubation. Working Stocks: 1xHDAC Assay Buffer: 50 mM HEPES (GIBCO 15630-114), 100mM KCl, 0.001% Tween-20 (Zymed 00-3005), 0.005% BSA (Invitrogen, P2489), pH 7.4, 1.5xHDAC+1.5xTrypsin: 225nM Trypsin(Worthinton)+0.2 ug/ml HDAC2 (BPS Inc) in assay buffer 300x Trypsin stock is made up in HDAC Assay Buffer, aliquoted and stored at -80C. 3xSubstrate: 13.8uM Substrate in assay buffer BPS Enzyme and Substrate amounts (per well).
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 302.4, ALogP 3.5, HBA 4, HBD 2, PSA 64.35, QED 0.85
Compound (SMILES): Cc1cn(C(=O)c2c(-c3c(Cl)cc(Cl)cc3Cl)noc2C(F)(F)F)c2cccc(/C=C/C(=O)O)c12
InChIKey: FPKKACLXGYDUPX-AATRIKPKSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Inhibitory Activity Against IDH1R132H and IDH1R132C Enzymes: The IDH1R132H protein and the IDH1R132C protein were prepared as follows: the IDH1R132H or IDH1R132C gene was integrated into a pET24b(+) vector (Novagen) to prepare a construct for C-terminal fusion of 6× histidine tag. After transformation of Rosetta 2 (DE3) E. coli, the expression of the protein was induced with IPTG. The E. coli was collected and homogenized, followed by the affinity purification of the 6× histidine fusion protein using HisTrap HP columns (GE Healthcare Japan Corp.) and gel filtration purification using Superdex 200 columns (GE Healthcare Japan Corp.) to obtain the IDH1R132H or IDH1R132C protein of interest.IDH1R132H and IDH1R132C each convert 2-oxoglutarate and NADPH to D-2-hydroxyglutarate (2-HG) and NADP+. Therefore, the activity of the IDH1R132H and IDH1R132C enzymes can be measured by detecting NADPH levels.The enzyme inhibitory activity evaluation was carried out as follows: 40 μL each of reaction solutions containing different concentrations of each compound (100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 20 mM MgCl2, 0.5 mg/mL bovine serum albumin, 1 mM reduced glutathione, 40 μM NADPH, 0.5 mM 2-oxoglutarate, 0.5% dimethyl sulfoxide, 50000 to 0.128 nM compound, and 12 nM IDH1R132H or 10 nM IDH1R132C as the enzyme) was added to each well of 384-well plates (Greiner Bio One International GmbH, #781096) and incubated at room temperature. While NADPH-derived fluorescence was occasionally monitored, the reaction was terminated by the addition of 5 μL of 0.5 M EDTA before consumption of NADPH. 5 μL of WST-8 reagent (Dojindo Laboratories, #CK04) was further added and mixed therewith. 15 minutes later, the absorbance at 450 nm was measured using a plate reader (PerkinElmer, Inc., EnVision). The observed absorbance value reflects the amount of residual NADPH. From the absorbance data, the enzyme inhibitory activity of each compound at each concentration was calculated and analyzed using medical statistical analysis software GraphPad Prism to calculate an IC50 value.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 543.71, ALogP 7.37, HBA 5, HBD 1, PSA 85.33, QED 0.27
Compound (SMILES): C=CC(=O)NC1CN(c2nc(Nc3cnn(C)c3)c3c(c2F)CNC3=O)C1
InChIKey: ITBDCDSJOWNJQS-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Kinase Inhibition Assay: EGFR: The working concentration of each component in 10 μl wild-type EGFR kinase reaction system was: 10 μM ATP, 0.8 ng/μl wild-type EGFR kinase (Invitrogen, PV3872), 2 μM Tyr04 substrate (Invitrogen, PV3193). After the compounds to be tested were added, the final concentration of DMSO was 2%. 10 mM drug stock solutions dissolved at room temperature were gradiently diluted with 4% DMSO in water to a final concentrations of 10-0.005 μM. To each well were added 2.5 μl of a solution of the test compounds and 5 μl of a mixture of wild-type EGFR kinase and Tyr04 substrate diluted by a reaction buffer, and then 2.5 μl of ATP was added to initiate the reaction. Reaction buffer instead of ATP were added to C1 wells, no drugs were added to C2 wells, and the phosphorylated substrates were added to C3 wells according to the instruction. After shaking on a shaker at 25° C. for 60 minutes in the dark, 5 μl of Development Reagent B (Invitrogen, diluted with TR-FRET dilution buffer) was added, and reacted on a shaking table at room temperature for 60 min. The plates were read in a VictorX5 fluorescent microplate reader (PerkinElmer) and the light absorbance at an excitation wavelength of 405 nm and emission wavelengths of 450 nm and 520 nm was measured (For example, C3520nm represents the reading of C3 well at 520 nm).
Measurement: IC50 = 1124 nM (pChEMBL 5.949999809265137)
Descriptors: MW 371.38, ALogP 0.43, HBA 7, HBD 3, PSA 104.18, QED 0.66
Compound (SMILES): COc1ccccc1-c1nnc([C@@H]2CCCN2C(=O)c2cc(C)c(C)cc2-n2nccn2)[nH]1
InChIKey: OWTICHBQSWXRQS-IBGZPJMESA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 uL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 uM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement.Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 uL/well, incubated for 120 min and finally 10 uL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist. The IC50 value (the concentration of compound needed to inhibit 50% of the agonistic response) is determined and may be normalized using the obtained IC50 value of an on-plate reference compound. Optimized conditions were achieved by adjustment of pipetting speed and cell splitting regime. The calculated IC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. In the case where IC50 values have been determined several times for the same compound, the geometric mean has been given.
Measurement: IC50 = 69 nM (pChEMBL 7.159999847412109)
Descriptors: MW 443.51, ALogP 3.66, HBA 7, HBD 1, PSA 101.82, QED 0.51
Compound (SMILES): Cc1ccc(NC(=O)c2ccnc(S(C)(=O)=O)c2)cc1-c1cnc(OC(C)C)c(N2CCOCC2)c1
InChIKey: ZJLHDNUYXKAUGC-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Raf Activity: Raf and biotinylated Mek, kinase dead, were combined at 2× final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40× of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2×ATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 0.21 nM (pChEMBL 9.680000305175781)
Descriptors: MW 510.62, ALogP 3.73, HBA 8, HBD 1, PSA 110.72, QED 0.51
Compound (SMILES): C=CC(=O)N(C)c1ncc2cc(-c3c(Cl)cccc3Cl)ccc2n1
InChIKey: RYYWVOZQTUXXHV-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: FGFR-1 wild type assay: In each well of a 384-well plate, 0.1 ng/ul of wild type FGFR-1 (Carna Biosciences, Inc.) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM CSKtide (5-FAM-KKKKEEIYFFFG-NH2) and 400 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35 s).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 358.23, ALogP 4.75, HBA 3, HBD 0, PSA 46.09, QED 0.63
Compound (SMILES): COc1cc2ncnc(N3CCN(c4ncc(OCc5cccc(C)c5)cn4)CC3)c2cc1OC
InChIKey: BAFRFOMJINMBBT-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Biochemical Activity Assay: Kit D816V assay: In each well of a 384-well plate, 0.04 ng/ul (0.5 nM) of D816V Kit (Carna Bioscience 08-156) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM Srctide (5-FAM-GEEPLYWSFPAKKK-NH2) and 15 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35 s). Data was normalized to 0% and 100% inhibition controls and the IC50 or EC50 calculated using a 4-parameter fit using GraphPad Prism.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 472.55, ALogP 3.65, HBA 9, HBD 0, PSA 85.73, QED 0.4
Compound (SMILES): Cc1c(C#N)ccnc1O[C@@H]1CC[C@@H](C)N(C(=O)c2cc(F)cnc2-c2nccs2)C1
InChIKey: VEEQAPLZSRNWFQ-CXAGYDPISA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Radioligand binding assay: Radioligand binding assay (described in Bergman et. al. Bioorg. Med. Chem. Lett. 2008, 18, 1425-1430) in which the inhibition constant (Ki) is determined in membranes prepared from CHO cells expressing either the OX1 or OX2 receptor. The intrinsic orexin receptor antagonist activity of a compound which may be used in the present invention may be determined by these assays.
Measurement: Ki = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 437.5, ALogP 3.99, HBA 7, HBD 0, PSA 92, QED 0.61
Compound (SMILES): CCCNC1CC12CCc1ccccc12
InChIKey: XSONEVCZKXRSDS-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biochemical Assay for LSD1 Activity (50 minutes): LSD1 demethylase reactions were carried out in 50 mM HEPES pH 7.4, 100 mM NaCl, 1 mM DTT, 0.01% Tween-20, and 0.1 mg/mL BSA. All enzymatic reactions were performed for either 50 minutes at room temperature in a 10-μL volume. Five microliters of 800 nM biotinylated H3K4me1 peptide solution was added to each well of a black 384 well Proxiplate containing 80 nL compound (final concentration of 0.8% DMSO). Reactions were initiated by the addition of a mixture containing 20 nM LSD1 and 20 nM FAD (5 μL). Enzyme activity was stopped by the addition of 5 μL of high salt buffer consisting of 50 mM HEPES pH 7.4, 1.5 M NaCl, 1 mM DTT, 0.01% Tween-20, and 0.1 mg/mL BSA. Capture of the product peptide by the anti-H3K4me0 antibody and Streptavidin APC was allowed to proceed for 60 mM at room temperature before measuring the TR-FRET signal. Europium-labeled antibody and Streptavidin APC were used at final concentrations of 0.003 nM and 100 nM, respectively (total assay volume of 20 μL). Plates were read on a Perkin Elmer EnVision. Percent inhibition was calculated using Max (no inhibitor) and Min (quenched with stop buffer) controls and inhibition curves plotted to determine IC50 values.
Measurement: IC50 = 529 nM (pChEMBL 6.28000020980835)
Descriptors: MW 201.31, ALogP 2.64, HBA 1, HBD 1, PSA 12.03, QED 0.79
Compound (SMILES): O=C(O)C1=C(C(=O)Nc2sc3c(c2-c2nc(C4CC4)ns2)CCCC3)CCCC1
InChIKey: KQVOAHIXLYQOEI-UHFFFAOYSA-N
Target: Fatty acid-binding protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q01469
Assay: Time Resolved-Fluorescence Energy Transfer (TR-FRET) Assay: Compounds were profiled for activity against human FABP4 (huFABP4) and/or human FABP5 (huFABP5) in Terbium (Tb) time resolved-fluorescence energy transfer (TR-FRET) assays monitoring the direct binding of Bodipy labeled fatty acid to His6 tagged FABP proteins (huFABP4 was expressed in house in E. Coli and purified, huFABP5 was purchased from Cayman Chemical Co., cat.no. 10010364), bound to Terbium labeled anti His6 tag antibody. Assay read-outs reflected energy transfer, upon binding of the ligand to the FABP protein, from the Terbium donor molecule to the acceptor Bodipy moiety. Final ligand concentration (125 nM) approximated the Kd for each protein.Stock DMSO solutions (1.8 mM) of compounds were serially diluted 3-fold for ten concentrations with 100% DMSO (50 μM to 0.003 μM final compound concentration). 1 μl of these compound dilutions and 1 μl of Bodipy labeled fatty acid 4.5 μM in 100% DMSO (Bodipy FL C11, cat. no. D3862, Invitrogen) were sequentially pipetted in wells of 384-well black polypropylene plates (Thermo Matrix cat. no. 4344). FABP4 or FABP5 protein was then added (28 μl of 64 nM protein in 25 mM Tris pH 7.5, 0.4 mg/ml γ-globulin, 1 mM DTT, 0.012% NP40, final protein concentration: 50 nM). Assay blanks contained ligand, but no protein. Neutral controls contained ligand, but no compound. After adding the detection reagent (Tb antiHis6 antibody, Columbia Biosciences, TB-110, 6 μl of a 24 nM Ab solution in 25 mM Tris pH 7.5, 0.4 mg/ml γ-globulin, final Tb antiHis6 Ab concentration: 4 nM), plates were spun one minute at 1000 rpm. Following an incubation at room temperature with shaking for 30 minutes, plates were read using an Envision reader (Perkin Elmer, Extinction wavelength: 340 nm, Emission: 490 nm and 520 nm, time delay: 100 μs; time window: 200 μs, 50 flashes).Final assay conditions were: 50 nM FABP protein, 125 nM Bodipy labeled fatty acid, 0.009% (vol/vol) NP40, 5.5% (vol/vol) DMSO in a total final assay volume of 36 μl. The assay was performed in triplicate.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 429.57, ALogP 4.92, HBA 6, HBD 2, PSA 92.18, QED 0.7
Compound (SMILES): COC[C@@H](C)Oc1cc(NC(=O)N2CCCc3cc(CN(C)C(=O)[C@H]4CCCO4)c(C=O)nc32)ncc1C#N
InChIKey: ATFNGRMKPANMIR-VGOFRKELSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymologic Experiment of FGFR1: In this experiment, the inhibitory effect of the compounds on FGFR1 kinase activity was tested by a fluorescence resonance energy transfer (TR-FRET) method, and the half maximal inhibitory concentration (IC50) of the compounds on the FGFR1 kinase activity was determined. 1) 15 μL of FGFR1 enzymatic solution was added to a 384-well plate, and the final concentration of the enzyme was 0.15 nM. 2) 15 μL of diluted solution in gradient of the compound was added.3) 15 μL of a substrate mixture containing substrate polypeptide with a final concentration of 550 nM and ATP with a final concentration of 10200 μM was added. 4) The mixture was incubated for 0.5-3 hours at room temperature. 5) 10 μL of EDTA and a test solution comprising labeled antibody were added, and the plate was incubated for 1 hour at room temperature. 6) The fluorescence signal values of each plate were determined by a microplate reader at 665 nm. 7) The inhibition rates were calculated according to the fluorescence signal values.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 536.59, ALogP 2.7, HBA 9, HBD 1, PSA 146.98, QED 0.48
Compound (SMILES): CCNC(=O)c1noc(-c2cc(Cl)c(O)cc2O)c1-c1cccc(CN2CCN(C(C)=O)CC2)c1
InChIKey: XQXUVHNMUXVLDZ-UHFFFAOYSA-N
Target: ATP-dependent molecular chaperone HSP82 (SINGLE PROTEIN), Saccharomyces cerevisiae S288c
UniProt: P02829
Assay: Fluorescence Polarization Assay: 1) Costar 96-well black assay plate. Assay buffer of (a) 100 mM Tris pH7.4; (b) 20 mM KCl; (c) 6 mM MgCl2. Stored at room temperature.3) BSA (bovine serum albumen) 10 mg/ml (New England Biolabs # B9001S)4) 20 mM probe in 100% DMSO stock concentration. Stored in the dark at RT. Working concentration is 200 nM diluted in AR water and stored at 4Â° C. Final concentration in assay 80 nM.5) E. coli expressed human full-length HSP90 protein, purified>95% (see, e.g., Panaretou et al., 1998) and stored in 50 uL aliquots at C. Protocol: 1) Add 100 ul buffer to wells 11A and 12A (=FP BLNK)2) Prepare assay reagents are kept on ice with a lid on the bucket as the probe is light-sensitive. i. Final Conc Hsp90 FP Buffer 10 ml 1xBSA 10 mg/ml (NEB) 5.0 ul 5 ug/ml Probe 200 uM 4.0 ul 80 nM Human full-length Hsp90 6.25 ul 200 nM 3) Aliquot 100 ul assay mix to all other wells 4) Seal plate and leave in dark at room temp for 20 minutes to equilibrate. Compound Dilution Plate 1x3 Dilution Series1) In a clear 96-well v-bottom plate {# VWR 007/008/257} add 10 ul 100% DMSO to wells B1 to H112) To wells A1 to A11 add 17.5 ul 100% DMSO 3) Add 2.5 ul cpd to A1. This gives 2.5 mM {50x} stock cpd assuming cpds 20 mM. 4) Repeat for wells A2 to A10. Control in columns 11 and 12.5) Transfer 5 ul from row A to row B-not column 12. Mix well.6) Transfer 5 ul from row B to row C. Mix well.7) Repeat to row G. 8) Do not add any compound to row H-this is the 0 row. 9) This produces a 1x3 dilution series from 50 uM to 0.07 uM.10) In well B12 prepare 20 ul of 100 uM standard compound. 11) After first incubation the assay plate is read on a Fusion a-FP plate reader. 12) After the first read, 2 ul of diluted compound is added to each well for columns 1 to 10. In column 11 {provides standard curve} only add compound B11-H11. Add 2 ul of 100 mM standard cpd to wells B12-H12 {is positive control} 13) The Zâ factor is calculated from zero controls and positive wells. It typically gives a value of 0.7-0.9.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 498.97, ALogP 3.49, HBA 7, HBD 3, PSA 119.14, QED 0.48
Compound (SMILES): C[C@H](NC(=O)[C@@H](CCc1ccccc1)NC1CCCC1)C(=O)NCc1ccc(C(=N)N)cc1
InChIKey: JRUBSEZNDNPDGW-FDDCHVKYSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: Enzymatic Assay for Thrombin: The thrombin assay utilizes a fluorogenic peptide substrate (Boc-VPR-AMC (R&D Systems) and was run at room temperature in an assay buffer containing 20 mM Hepes, pH 7.4, 140 mM NaCl and 0.1% Tween 20. Assay parameters were adjusted such that the assay was linear with respect to time, enzyme and substrate concentrations. Under these optimized assays conditions, IC50values were equivalent to Ki values, except in a few cases of tight binding inhibitors. Cases of tight binding or possible slow binding inhibitors were handled by the methods described in Copeland R. A. (2013) Evaluation of Enzyme Inhibitors in Drug Discovery. 2nd Ed. John Wiley and Sons, Inc., Chapters 5-7.
Measurement: Ki < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 449.6, ALogP 2.63, HBA 4, HBD 5, PSA 120.1, QED 0.27
Compound (SMILES): CN1C(=O)[C@@H](NC(=O)c2nnc(Cc3cccc(C#N)c3)[nH]2)COc2cccnc21
InChIKey: LGWRJODXWBECFU-AWEZNQCLSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Fluorescent Polarization Binding Assay: Fluorescent polarization binding assay was performed in polystyrene low volume 384-well black plate, at Room Temperature (RT) in a final volume of 10.1 μl/well using 10 nM of GST-hRIPK1 (8-327) enzyme and 5 nM of fluorescent-labeled ligand (14-(2-{[3-({2-{[4-(cyanomethyl)phenyl]amino}-6-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]-4-pyrimidinyl}amino) propyl]amino}-2-oxoethyl)-16,16,18,18-tetramethyl-6,7,7a,8a,9,10,16,18-octahydrobenzo[2″,3″ ]indolizino[8″,7″: 5′,6′]pyrano[3′,2′:3,4]pyrido[1,2-a]indol-5-ium-2-sulfonate.Test Compounds were serially diluted in DMSO at 100 fold final concentrations in the assay (1% DMSO final). In each well of a 384-well Plate were dispensed 0.1 μL of compound solution (or DMSO for controls) followed by 5 μL of GST-hRIPK1 (8-327) at twice the final concentrations in assay buffer (50 mM HEPES pH 7.5, 10 mM NaCl, 50 mM MgCl2, 0.02% CHAPS, 0.5 mM DTT and 0.01% Pluronic F127). For negative control the enzyme addition was replaced by assay buffer only.After addition of 5 μL of fluorescent-labeled ligand at twice the final concentrations in assay buffer, the plate was incubated at RT for 30 min. At the end, the binding was measured as FP value with the Envision (PerkinElmer) plate reader using filter for an excitation λ=531 nm FP and an emission λ=595 nm FP (S & P-pol).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 403.4, ALogP 0.82, HBA 7, HBD 2, PSA 136.89, QED 0.66
Compound (SMILES): Cc1c(N(C)[C@H]2CCN(Cc3ccccc3)C2)cnc(S(=O)(=O)Nc2cccc(F)n2)c1F
InChIKey: RLTVQPTYWXWWPV-SFHVURJKSA-N
Target: Sodium channel protein type 1 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35498
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 30000 nM (pChEMBL 4.519999980926514)
Descriptors: MW 473.55, ALogP 3.57, HBA 6, HBD 1, PSA 78.43, QED 0.53
Compound (SMILES): Cc1ccc(C(O)(C(N)=O)C(F)(F)F)cc1-c1cnc(N)c(N2CCN(C(=O)C3CC3)CC2)n1
InChIKey: KTUGNJQDCJISGN-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI3Kdelta Scintillation Proximity Assay: Materials: [γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kδ (p110δ/p85α) Recombinant Human Protein was purchased from Eurofins (St Charles, Mo.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kδ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 3.4 nM PI3Kδ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (PerkinElmer). IC50 determination was performed by fitting the curve of percent of the solvent control activity versus the log of the inhibitor concentration using the GraphPad Prism 6.0 software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 478.48, ALogP 1.33, HBA 7, HBD 3, PSA 138.67, QED 0.59
Compound (SMILES): COC1CCN(c2cccc3c2CCN(C(=O)c2cnn(-c4cccc(Cl)c4)c2)C3C(=O)Nc2ccc(C(=O)O)cc2)CC1
InChIKey: RESLNCPFDIUSRS-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Enzyme Inhibition Assay: Plasma kallikrein determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.1-0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human plasma kallikrein (Enzyme Research Laboratories) at a final assay concentration of 200 pM and the synthetic substrate S-2302 (H-(D)-Pro-Phe-Arg-pNA; CHROMOGENIX) at a concentration of 0.00008-0.0004 M.
Measurement: Ki = 229 nM (pChEMBL 6.639999866485596)
Descriptors: MW 614.1, ALogP 5.22, HBA 7, HBD 2, PSA 117, QED 0.29
Compound (SMILES): Cc1ncc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-c1cc(N2CCOCC2)nc(N2CC(O)C2)n1
InChIKey: XBUYJNSUEWENMF-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Raf Activity: Raf and biotinylated Mek, kinase dead, were combined at 2× final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40× of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2×ATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 514.51, ALogP 3.14, HBA 8, HBD 2, PSA 103.71, QED 0.54
Compound (SMILES): O=C1CCC(c2ccccc2)N1c1ccc(-c2noc(C(F)(F)F)n2)cc1F
InChIKey: WEWDMCBZNJFRKL-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Enzymatic Assay: HDAC1 and 6 reagents: FLAG-tagged HDACs 1 and 6 were prepared in-house by protein expression in HEK293F cells followed by anti-FLAG affinity purification. Assays were performed with buffer containing 20 mM HEPES, pH 8.0 [Boston BioProducts, catalog #BB-104, 1M stock], 137 mM NaCl [Sigma, catalog #S5150, 5M stock], 2.7 mM KCl [BioChemika, catalog #87526, 4M stock], 1 mM MgCl2 [Fluka, catalog #63020, 1M stock], and 0.05% BSA (Fraction V) [Invitrogen, catalog #15260, 7.5% stock]. In addition to the above buffer ingredients, TCEP [CalBiochem, catalog #580561, 500 mM stock] was added at a final concentration of 0.5 mM to the buffer for the HDAC6 assays. HDAC1, 2, 3, and 6 enzymes were run at the final concentrations of 0.3 nM, 1.5 nM, 0.3 nM, and 1.333 nM, respectively. Fluor-de-Lys substrate [BioMol Research Laboratories, catalog #KI-104], used to evaluate enzyme activity, was added at the final concentrations of 20 uM, 40 uM, 20 uM, and 2.5 uM for HDACs 1, 2, 3, and 6. To enable detection of the signal, Developer [BioMol Research Laboratories, catalog #KI-105] was added at a 1:250 dilution to the stop solution, which also included 10 uM SAHA [Sigma, catalog #SML0061] to ensure complete termination of the reaction.
Measurement: IC50 = 530 nM (pChEMBL 6.28000020980835)
Descriptors: MW 391.32, ALogP 4.76, HBA 4, HBD 0, PSA 59.23, QED 0.6
Compound (SMILES): Nc1cc(C(CCCc2ccc3ccccc3c2)c2cnn(Cc3ccc4c(c3)CCNC4)c2)c2nn[nH]c2n1
InChIKey: WDIGQGKTBMRYBB-UHFFFAOYSA-N
Target: Myeloperoxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P05164
Assay: MPO Chlorination Assay (APF Assay): MPO chlorination activity was measured in 100 mM KPi (pH 7.4) by utilizing the non-fluorescent reagent Aminophenyl fluorescein (APF, Invitrogen Cat. #A36003). APF is cleaved by (OCl) to yield the fluorescent compound fluorescein. Reactions were carried out in 50 μL total volume by adding a 25 μL mixture of 200 pM myeloperoxidase and 120 mM NaCl to 100 nL inhibitor in 100% DMSO in a 384 well, non-binding surface clear bottom plate (Corning #3655). Enzyme, inhibitor, and chloride were preincubated for ten minutes at rt.After the ten minute preincubation, 25 μL of an APF mixture containing 10 mM APF, 120 mM NaCl and 10 μM H2O2 was added to the plate using the internal dispensing system of a Hammatsu FDSS 6000. Kinetic determinations were carried out immediately on the FDSS 6000 (3 minute kinetic read, 1 read every second, ex: 485 nm, em: 535 nm). IC50 values for inhibitors were calculated by taking the slope of the linear portion of the kinetic measurement (20 seconds to 80-120 secs).IC50 values were calculated by determining the slope of the linear portion of the kinetic trace (180-540 secs), and using that calculated slope to determine % inhibition occurring at each concentration of inhibitor using the following equation:Y = A + B - A 1 + ( C / x ) Dwhere A=minimal Y value (activity level of inhibited sample), B=maximal Y value (activity level of uninhibited sample), C=Log IC50, D=Hill Slope, x=concentration of inhibitor.The exemplified Examples disclosed below were tested in the MPO peroxidation assay described above and found to have MPO inhibitory activity. A range of IC50 values of ≤10 μM (10000 nM) was observed.
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 528.66, ALogP 5.13, HBA 7, HBD 3, PSA 110.33, QED 0.25
Compound (SMILES): CC(C)[C@@H]1CN(c2ccc(F)c(S(C)(=O)=O)c2)CCN1c1ncc(Cc2ccc(OC=O)cc2)c(C(F)(F)F)n1
InChIKey: CRBFBKFQHKHSNV-QHCPKHFHSA-N
Target: Oxysterols receptor LXR-beta (SINGLE PROTEIN), Homo sapiens
UniProt: P55055
Assay: LXR alpha/beta Radioligand Binding Assay: Compounds of the invention were assessed in a competition binding assay where different concentrations of compounds were incubated with the LXR ligand binding domain (LBD) in the presence of radiolabeled LXR ligand [3H]TO901317. The amount of the LXR-LBD that complexed with [3H]T0901317 was measured by scintillation proximity assay (SPA) employing non-specific binding of LXR-LBD to poly-lysine coated Yttrium silicate beads. Partially purified LXR α or β LBD protein (15-45 nM) was incubated at rt for 30 min with 15 nM [3H]TO901317 (25-40 Ci/mmol) and different concentrations of test compounds in 80 μL of phosphate buffered saline (PBS) buffer containing 2.5% DMSO, 1% glycerol, 2 mM EDTA, 2 mM CHAPS and 5 mM DTT in 96-well plates. Poly-lysine SPA beads (50 μg) were added to each well and the total volume was adjusted to 120 μL. The plates were shaken on an orbital shaker for 20 min and then allowed to settle for 10 more min at rt before a brief centrifugation at 2,000 rpm for 1 min. The SPA signal was measured on a MicroBeta® liquid scintillation counter (Perkin Elmer, Waltham, Mass.), and the results were used for calculating IC50 values based on the total binding (DMSO control) and non-specific binding (5 μM of unlabeled TO901317) controls. The Ki values were calculated according to equation 1, where [RL] is the final concentration of [3H]TO901317 in the assay, and the Kd values of 20 nM and 10 nM of TO901317 for LBDs of LXRα and LXRβ, respectively, were determined by direct titration of the radioligand with these proteins.
Measurement: Ki = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 580.6, ALogP 4.52, HBA 8, HBD 0, PSA 92.7, QED 0.28
Compound (SMILES): N#Cc1cnc(NC(=O)N2CCCc3cc(CN4CCOCC4=O)c(C=O)nc32)cc1NCCOC1CCCC1
InChIKey: OKNAPKMOEKWABB-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymologic Experiment of FGFR1: In this experiment, the inhibitory effect of the compounds on FGFR1 kinase activity was tested by a fluorescence resonance energy transfer (TR-FRET) method, and the half maximal inhibitory concentration (IC50) of the compounds on the FGFR1 kinase activity was determined. 1) 15 μL of FGFR1 enzyme solution was added to a 384-well plate, and the final concentration of the enzyme was 0.15 nM. 2) 15 μL of diluted solution in gradient of the compound was added. 3) 15 μL of a substrate mixture containing substrate polypeptide with a final concentration of 550 nM and ATP with a final concentration of 10200 μM was added. 4) The mixture was incubated for 0.53 hours at room temperature. 5) 10 μL of EDTA and a test solution comprising labeled antibody were added, and the plate was incubated for 1 hour at room temperature. 6) The fluorescence signal values of each plate were determined by a microplate reader at 665 nm.7) The inhibition rates were calculated according to the fluorescence signal values.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 547.62, ALogP 2.88, HBA 9, HBD 2, PSA 149.78, QED 0.36
Compound (SMILES): CNS(=O)(=O)N1CC(CC#N)(n2ccc(-c3nc(-c4cnn(C)c4)cc4ncccc34)n2)C1
InChIKey: FLAGMGVIPOZBLE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1mM ATP: 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 = 2369 nM (pChEMBL 5.619999885559082)
Descriptors: MW 463.53, ALogP 1.28, HBA 9, HBD 1, PSA 134.62, QED 0.46
Compound (SMILES): Clc1cccc(-c2nc3c(c(Nc4ccc(Cc5nn[nH]n5)cc4)n2)SCCC3)c1
InChIKey: AYTYFOCXTDAXDZ-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: Kinetic Assay: Assays are performed in 96-well plates in a total volume of 200 μl/well. Compounds are dissolved in dimethylsulfoxide (DMSO) and added to plates in a volume of 10 μl followed by addition of 165 μl of assay mix. Plates are pre-incubated at 25° C. for 15 min and the reactions are initiated by the addition of 25 μl of cAMP followed by thorough mixing. Reaction rates are measured by monitoring the decrease in fluorescence using excitation at 355 nm and emission at 460 nm for a period of 10 min in a fluorescence plate reader. Initial rates (slopes) are determined from linear portions of the progress curves. Final concentrations of assay components are as follows: 50 mM Tris, pH 8, 10 mM MgCl2, 50 mM KCl, 2% DMSO, 5 mM tris(2-carboxyethyl)phosphine (TCEP), 0.4 mM phosphenolpyruvate (PEP), 0.01 mM NADH, 0.04 mM adenosine triphosphate (ATP), 0.004 mM cAMP, 7.5 units myokinase from yeast, 1.6 units pyruvate kinase, 2 units lactate dehydrogenase, and either 0.5 nM human PDE4D7 or 10 nM human PDE4B1. All data are percent normalized relative to controls and are presented as percent inhibition. An inhibitory concentration 50% (IC50) value is calculated by fitting of a sigmoidal dose response curve. Human PDE4D7 contained a mutation of serine 54 to aspartic acid to mimic activation by cAMP-dependent protein kinase A (PKA). Human PDE4B1 contained a corresponding mutation of serine 133 to aspartic acid to also mimic PKA activation.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 435.94, ALogP 4.68, HBA 7, HBD 2, PSA 92.27, QED 0.47
Compound (SMILES): NC(=O)c1cc(Nc2nc(-c3cccc(C(F)(F)F)n3)nn3cccc23)ccn1
InChIKey: LARHPAPBTVOUHP-UHFFFAOYSA-N
Target: TGF-beta receptor type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P37173
Assay: Biological Assays: Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 > 15000 nM (pChEMBL 4.823999881744385)
Descriptors: MW 399.34, ALogP 3.05, HBA 7, HBD 2, PSA 111.09, QED 0.55
Compound (SMILES): CCCCCCCCc1cccc(-c2noc(C[C@@H]3CCCN3C(=N)N)n2)c1
InChIKey: ASNASFWTSOQODI-IBGZPJMESA-N
Target: Sphingosine kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NRA0
Assay: Scintillation Proximity Assay: SphK activity was measured by a scintillation proximity assay as previously described [1], the disclosure of which is incorporated herein. Briefly, recombinant SphK1 or SphK2 were expressed in Sf9 insect cells, crude homogenates were prepared and incubated in 96 well FlashPlates (Perkin-Elmer) in a buffer containing D-erythro-sphingosine and γ-[33P]ATP. The [33P]S1P product, which adheres to the plate wall, was quantified by scintillation counting. To assay ceramide kinase or diacylglycerol kinases, the recombinant proteins were incubated with γ-[32P]ATP and substrate (C6 ceramide or 1-Ohexadecyl-2-acetyl-sn-glycerol, respectively) and the lipid product, after recovery by organic extraction, was resolved by thin layer chromatography, detected by autoradiography, and quantified by liquid scintillation counting. These assays were performed with and without a fixed concentration of inhibitor and its effect on KM and Vmax determined.
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 383.54, ALogP 4.54, HBA 4, HBD 2, PSA 92.03, QED 0.36
Compound (SMILES): CC1(C)CCCN1S(=O)(=O)c1ccc(Nc2nn([C@]3(CC#N)CCCOC3)c3cc[nH]c(=O)c23)cc1
InChIKey: VVZVBFLIZOTMLJ-VWLOTQADSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay : The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 0.33 nM (pChEMBL 9.479999542236328)
Descriptors: MW 510.62, ALogP 3.45, HBA 8, HBD 2, PSA 133.11, QED 0.52
Compound (SMILES): Cc1cc(Cl)ccc1-n1c(C(=O)NC2CC2)nc2cccnc21
InChIKey: QDXDJUKHXSVGLR-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: Scintillation Proximity Assay (SPA): The PDE4B and 4D assays use scintillation proximity assay (SPA) technology to measure the inhibition of human recombinant PDE4B and PDE4D enzyme activity by compounds in vitro. The PDE4B and 4D assays are run in parallel using identical parameters, except for the concentration of enzyme (32 pM PDE4B and 16 pM PDE4D). The assays are performed in a 384-well format with 50 μL assay buffer (50 mM Tris pH 7.5; 1.3 mM MgCl2; 0.01% Brij) containing enough PDE4B and PDE4D to convert 20% of substrate (1 μM cAMP consisting of 20 nM 3H-cAMP+980 μM cold cAMP) and a range of inhibitors. Reactions are incubated for 30 min at 25° C. The addition of 20 μL of 8 mg/mL yitrium silicate SPA beads (PerkinElmer) stops the reaction. The plates are sealed (TopSeal, PerkinElmer) and the beads are allowed to settle for 8 hrs, after which they are read on the Trilux Microbeta overnight.
Measurement: IC50 > 17900 nM (pChEMBL 4.747000217437744)
Descriptors: MW 326.79, ALogP 3.27, HBA 4, HBD 1, PSA 59.81, QED 0.8
Compound (SMILES): C[C@H](NC(=O)c1c(N)ncn2ccnc12)c1cc2cccc(Cl)c2c(=O)n1-c1ccccc1
InChIKey: ZRDSSDUSMNALEF-AWEZNQCLSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
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 458.91, ALogP 3.76, HBA 7, HBD 2, PSA 107.31, QED 0.43
Compound (SMILES): Nc1nn2cc(Cl)cnc2c1C(=O)Nc1cnccc1N1CCn2cncc2C1
InChIKey: SOZLBTJNUOAWQC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Table 5: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 μM [y-33P]ATP (3 mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide(ASELPASQPQPFSAKKK) (SEQ ID NO:1).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).
Measurement: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 409.84, ALogP 1.83, HBA 9, HBD 2, PSA 119.26, QED 0.53