lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccc(S(=O)(=O)N2[C@@H]3CCC[C@H]2C[C@H](Nc2nc(Nc4cc(C)[nH]n4)cc4ncccc24)C3)cn1
InChIKey: JFRIPMLOQQWZKX-OWZKRDHZSA-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 518.65, ALogP 4.29, HBA 8, HBD 3, PSA 128.79, QED 0.34
Compound (SMILES): CN(C(=O)Nc1cc(Cl)c(C#N)cn1)c1ccc(CO)c(C=O)n1
InChIKey: WMRIFGQWUKMDIF-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Inhibition Assay: FGFR2: The in vitro activity of FGFR2 was determined by assaying the phosphorylation level of the substrate in the kinase reaction, by means of an HTRF kinase assay kit. The reaction buffer comprised the following components: 5-fold diluted Enzymatic buffer/kinase 5× (main ingredient: 50 mM HEPES, pH 7.0), 5 mM MgCl2, 1 mM DTT; the human recombinant FGFR2 catalytic structural domain protein (amino acids 400-821) was commercially available from Beijing Sino Biological Inc., diluted with the reaction buffer to a 0.045 ng/μL kinase solution; the substrate reaction solution comprised a biotin labeled tyrosine kinase substrate diluted with the reaction buffer to 800 nM, and 50 μM ATP, and the assay solution comprised an Eu3+ labeled cage-shaped antibody (Cisbio, Catalog number 61T66KLB) diluted with the assay buffer (Cisbio, Catalog number 62SDBRDF) to 0.125 ng/μL, and 50 nM streptavidin labeled XL665.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 345.75, ALogP 1.97, HBA 6, HBD 2, PSA 119.21, QED 0.82
Compound (SMILES): Cn1ncc(-c2cc(-c3cnn(C4(CC#N)CC(C#N)C4)n3)c3cccnc3c2)c1CO
InChIKey: WFAVUGXESLKHMC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 = 8630 nM (pChEMBL 5.059999942779541)
Descriptors: MW 424.47, ALogP 2.93, HBA 9, HBD 1, PSA 129.23, QED 0.52
Compound (SMILES): O=C(CN1C(=O)CCc2ccccc21)Nc1ccsc1-c1c[nH]cn1
InChIKey: GFWLYZCXFSXTMS-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P45984
Assay: HTRF Assay: (1) Assay buffer #1 with 0.0025% Tween and 1 mM DTT was prepared using JNK buffer stock solution.(2) Assay buffer #2 with 0.025% BSA was prepared using the assay buffer #1.(3) Test compound solution preparation: 5× compound solution was prepared using assay buffer #1 with 5% DMSO. The compound solution (10 μl/well) was added to a 384-well plate (Corning, Cat No. 3654).(4) aJNK1, 2 or 3 preparation: aJNK stock (100 μg/ml) at −80° C. was thawed on ice and an aJNK (1.6 ng/ml) solution was prepared using assay buffer #2. The aJNK solution (20 μl/well) was added to the plate and the plate was shaken. The enzyme was incubated with the compound at RT for 10 min.(5) ATP/substrate solution preparation: ATP and substrate stocks were thawed on ice. 2.5×ATP/substrate (e.g., 25 μM or 2.5 mM ATP/50 nM EPIW-1) was prepared using assay buffer #1. The ATP/substrate (20 μl/well) was added to the plate and the plate was shaken. The plate was incubated at RT for 15 min. Note: In an exemplary assay, the final ATP concentration was about 1 mM.(6) EDTA preparation: 30 mM EDTA was prepared using 0.5M EDTA stock and assay buffer #1. The EDTA (10 μl/well) was added to the plate to quench the enzyme reaction and the plate was shaken well.(7) Detection reagent preparation: Eu-63 and SA-XL stocks were thawed on ice. 4× Eu-63/SA-XL (2 nM Eu-63/9.4 nM SA-XL) was prepared using JNK detection buffer. The Eu-63/SA-XL solution (20 μl/well) was added to the plate and the plate was shaken. The plate was incubated at RT for 1 hr before reading the plate on LJL using ratiometric method named HTRF.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 352.42, ALogP 3.06, HBA 4, HBD 2, PSA 78.09, QED 0.76
Compound (SMILES): CNC(=O)Nc1cn(C)c2c1C(=O)N(c1cc(C)c3nnn(C)c3c1)C2c1ccc(Cl)cc1
InChIKey: ZDYWXTGNQATUAC-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: Assay for CREB protein: All assays were performed in 384-well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Perkin Elmer/Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO HummingBird nanodispenser (Zinsser Analytic). The assay was started by stepwise addition of 4.5 uL per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 45 nM His-Brd2(60-472) or 45 nM His-Brd3(20-477) or 45 nM His-Brd4(44-477) all proteins produced in-house) and 4.5 uL per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 60 nM acetyl-histone H4 (AcK 5, 8, 12, 16) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 uL per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 3 nM Eu-labeled anti-His6 antibody, 21 nM streptavidin-allophycocyanin) were added. After 35 minutes incubation at 30° C., plates were measured in a Perkin Elmer EnVision multilabel reader.
Measurement: IC50 = 1300 nM (pChEMBL 5.889999866485596)
Descriptors: MW 463.93, ALogP 3.77, HBA 6, HBD 2, PSA 97.08, QED 0.48
Compound (SMILES): CN(c1ncnc2[nH]ccc12)C1C[C@@H]2CCN(C(=O)CC#N)C[C@@H]2C1
InChIKey: NRMPNHBYSBGAQE-RFHHWMCGSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro Kinase Assay: In vitro kinase assays described below can be used to determine the activity of a test compound for inhibiting the activity of JAK1 kinase. The test compounds were dissolved in dimethyl sulfoxide and diluted with water to a serial concentration gradient as required in the experiment. JAK1 substrates (Cell Signaling Technology, Catalog Number: 1305s) and ATP (2 mM) solution were diluted with water to obtain a final concentration of 20 uM ATP and 1.2 uM substrate solution. The appropriate amount of JAK1 kinase (Invitrogen, Catalog Number: pv4774) was mixed with 4x buffer (prepared by user, and comprising 50 mM HEPES, pH 7.3, 125 mM NaCl, 24 mM MgCl2, 1.25 mM DTT) to a final concentration of 8 ng/uL. To each well of a microplate [DELFIA Streptavidin-coated clear plate (Perkin Elmer, Catalog Number: AAAND-0005)]17.5 uL of ATP/substrate mixture, 5 uL of an aqueous solution of a test compound (5 uL of pure water only were added to the control and blank), and 7.5 uL of the kinase solution prepared above (4x buffer only was added to the control) were added. Each well was mixed sufficiently, then incubated at room temperature (27° C.) for 50 minutes, washed with wash buffer, and dried three times, then HRP conjugated antibody was added [Phospho-Tyrosine Mouse mAb (P-Tyr-100) (HRP Conjugate, Cell Signaling Technology, Catalog Number: 5465)], and incubated for 1 hour. The microplate was washed with wash buffer and dried three times, and then TMB (Sigma, Catalog Number: T4444) was added, and incubated for 5 to 15 minutes to allow for color change. Stop solution (1 N sulfuric acid solution) was added to stop the reaction. Absorbance was measured on a Novostar microplate reader at a wavelength of 450 nm. IC50 values of test compounds was calculated from the data of the test compounds in inhibiting the activity of JAK1 kinase at different concentrations.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 338.42, ALogP 1.93, HBA 5, HBD 1, PSA 88.91, QED 0.92
Compound (SMILES): Cc1c(Cl)cc(Cl)cc1N1CCN(C(=O)C(C)CC2(C)NC(=O)NC2=O)C[C@@H]1C
InChIKey: KUJHASPUCDKGOT-BIAXGUISSA-N
Target: 72 kDa type IV collagenase (SINGLE PROTEIN), Homo sapiens
UniProt: P08253
Assay: In Vitro Assay: MMP2: Protocol 2: The basis for the assay is the cleavage of the substrate 390 MMP FRET substrate I (Anaspec, Catalog n#: AS-27076) by human MMP2 (R&D SYSTEMS INC., Cat #902-MP).For the dose response (10 point), 4 μL of a dilution series of compound (2 mM highest concentration, 1/5 dilution in DMSO further diluted 1 in 10 in water, corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (45 mM Tris pH 7.5, 9 mM CaCl2, 135 mM NaCl, 0.045% Brij35) containing MMP2 (0.03 ng/L) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate 390 MMP FRET substrate I (10 μL, 2.5 μM, Anaspec) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 30 min at room temperature (Excitation 485 nm, Emission 530).
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 441.36, ALogP 2.96, HBA 4, HBD 2, PSA 81.75, QED 0.7
Compound (SMILES): COc1cc(F)cc(C2CCN(C(=O)CCC3(C)NC(=O)NC3=O)CC2)c1
InChIKey: OQTMQIXWHKKLMK-UHFFFAOYSA-N
Target: Collagenase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P45452
Assay: hMMP13 assay: The basis for the assay is the cleavage of the substrate 390 MMP FRET Substrate I (Anaspec Cat# AS-27076) by human MMP13 (Chemicon, Cat#CC068).For the dose response (10 point), 4 μL of a dilution series of compound (20 μM highest concentration, 1/5 dilution in water), is transferred to 384 well Fluotrac 200 plate (Greiner, cat#781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (50 mM Tris pH7.5, 150 mM NaCl, 10 mM CaCl2, 0.05% CHAPS, 5 μM ZnCl2) containing MMP13 (0.01 ng/μL) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration). Human MMP13 is preactivated by incubated the enzyme in the same buffer complemented with 1 mM freshly prepared p-Aminophenylmercuric acetate (AMPA) for 1 h at 37° C.The reaction is initiated by adding to the assay plate 390 MMP FRET Substrate I (10 μL, 2.5 μM) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 45 min at room temperature (Excitation 485 nm, Emission 530).
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 377.42, ALogP 1.92, HBA 4, HBD 2, PSA 87.74, QED 0.77
Compound (SMILES): Cc1nc(S(=O)(=O)Nc2cccc(F)n2)c(F)cc1NCc1ccccc1CN(C)C(C)(C)C
InChIKey: HAZWRLJBBBQVGU-UHFFFAOYSA-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 1-1.5h 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 CaC2, 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 = 16735 nM (pChEMBL 4.78000020980835)
Descriptors: MW 489.59, ALogP 4.71, HBA 6, HBD 2, PSA 87.22, QED 0.45
Compound (SMILES): CN(Cc1ccc2c(c1)CCC(C(=O)NO)=C2)CC12CC3CC(CC(C3)C1)C2
InChIKey: GOBRTECWEHXUDO-UHFFFAOYSA-N
Target: Histone deacetylase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P56524
Assay: HDAC Inhibition Assay: All histone deacetylases were purchased from BPS Bioscience. The substrates, Broad Substrate A, and Broad Substrate B, were synthesized and are now available commercially through Perkin Elmer. All the other reagents were purchased from Sigma-Aldrich. Caliper EZ reader II system was used to collect all data. HDAC inhibition assays: Compounds were tested in duplicate in a 12-point dose curve with 3-fold serial dilution starting from 33.33 μM. Purified HDACs were incubated with 2 μM carboxyfluorescein (FAM)-labeled acetylated or trifluoroacetylated peptide substrate (Broad Substrate A and B respectively) and test compound for 60 minutes at room temperature, in HDAC assay buffer that contained 50 mM HEPES (pH 7.4), 100 mM KCl, 0.01% BSA and 0.001% Tween-20. Reactions were terminated by the addition of the known pan HDAC inhibitor LBH-589 (panobinostat) with a final concentration of 1.5 μM. Substrate and product were separated electrophoretically and fluorescence intensity in the substrate and product peaks was determined and analyzed by Labchip EZ Reader. The reactions were performed in duplicate for each sample. IC50 values were automatically calculated by Origin 8 using 4 Parameter Logistic Model. The percent inhibition was plotted against the compound concentration, and the IC50 value was determined from the logistic dose-response curve fitting by Origin 8.0 software.
Measurement: IC50 = 389 nM (pChEMBL 6.409999847412109)
Descriptors: MW 380.53, ALogP 4.17, HBA 3, HBD 2, PSA 52.57, QED 0.6
Compound (SMILES): CCc1cc(O)c(F)cc1-c1cc2[nH]ncc2c(NCc2ccccc2N(CCO)S(C)(=O)=O)n1
InChIKey: UVUFBQMWXOMULF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.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 = 18.5 nM (pChEMBL 7.730000019073486)
Descriptors: MW 499.57, ALogP 3.4, HBA 7, HBD 4, PSA 131.44, QED 0.28
Compound (SMILES): CN1C(=O)C[C@@H](Cc2ccccc2)c2nnc3cc(-c4ccnc(Nc5ccnn5C)c4)cc1n23
InChIKey: FRGAEHIHCCSLPK-HXUWFJFHSA-N
Target: Mitogen-activated protein kinase 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16659
Assay: Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an n-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100× final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 2 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 μM S/T17 peptide substrate and 25 μM ATP (corresponding to the observed Km) for a total reaction volume of 25 μL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm. The data collected from each well was fit to a straight line, and the resulting rates were used to calculate percent of control. Percent of control was plotted against compound concentration, and IC50 values were determined using a four-parameter fit.
Measurement: IC50 = 0.073 nM (pChEMBL 10.140000343322754)
Descriptors: MW 464.53, ALogP 3.96, HBA 8, HBD 1, PSA 93.24, QED 0.42
Compound (SMILES): COc1cc(-c2[nH]c3ccc(C4CCN(CC(=O)N5C(C)CCC5C)CC4)cc3c2C(C)C)cn2ncnc12
InChIKey: GTLMMBIUPDQPAK-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 = 3913 nM (pChEMBL 5.409999847412109)
Descriptors: MW 528.7, ALogP 5.59, HBA 6, HBD 1, PSA 78.76, QED 0.35
Compound (SMILES): CC(C)n1nc(-c2cc3ccc(-c4ncco4)cc3[nH]2)c2c(N)ncnc21
InChIKey: HOZLFOUMPFCLFK-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Enzyme Assay: Kinase activity was detected using CisBio HTRF kinEASE kit based on time-resolved fluorescence transfer (FRET). The assay was performed in 384-well white plates (Corning #3574) in a reaction volume of 10 μL containing 1× CisBio enzymatic buffer supplemented with a final concentration of 5 mM MgCl2, 1 mM DTT, 10 nM SEB and 0.01% Triton X100 for RET. The same buffer conditions were used for KDR with the addition of 2 mM MnCl2. RETV804M buffer used 1× CisBio enzymatic buffer supplemented with a final concentration of 2 mM MgCl2, 1 mM DTT, 20 nM SEB and 0.01% Triton X100.
Measurement: IC50 = 8000 nM (pChEMBL 5.099999904632568)
Descriptors: MW 359.39, ALogP 3.79, HBA 7, HBD 2, PSA 111.44, QED 0.51
Compound (SMILES): N[C@@]1(C(=O)O)CNC[C@H]1CCCB(O)O
InChIKey: NDZPRHZISXYFRQ-SVRRBLITSA-N
Target: Arginase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P05089
Assay: Inhibition Assay: Inhibition of arginase I (ARG I) and arginase II (ARG II) by Formula I or Formula II compounds is followed spectrophotometrically at 530 nm. The compound to be tested was dissolved in DMSO at an initial concentration 50-fold greater than its final concentration in the cuvette. 10 μl of the stock solution was diluted in 90 μl of the assay buffer that comprises 0.1M sodium phosphate buffer containing 130 mM NaCl, pH 7.4, to which is added ovalbumin (OVA) at a concentration of 1 mg/ml. Solutions of arginase I and II were prepared in 100 mM sodium phosphate buffer, pH 7.4 containing 1 mg/ml of OVA to give an arginase stock solution at a final concentration of 100 ng/ml.
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 216.05
Compound (SMILES): C[C@](O)(CS(=O)(=O)c1ccc(F)cc1)C(=O)Nc1cnc(C#N)c(C(F)(F)F)c1
InChIKey: JWQMHMGGGRQTSY-INIZCTEOSA-N
Target: Androgen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P10275
Assay: Gal4-luciferase reporter assay: Briefly, the AR LBD is expressed as a fusion with the Gal4 transcription factor, which binds to the Gal4 reporter DNA. Upon activation with agonist hormone (DHT), the Gal-AR LBD binds to the Gal4 reporter gene, which in turn drives the transcription (and subsequently translation) of the reporter luciferase. The effect of antiandrogens (competitive antagonists) on this process is measured by quantifying the amount of luciferase activity after 24 hours in the presence of varying concentrations of antiandrogens. From these values, an IC50 for each antagonist is calculated. Experimentally, Hela cells were maintained in Dulbecco's modified Eagle's medium H-21 4.5 g/L glucose, containing 10% steroid depleted fetal bovine serum, 50 units/mL penicillin. For transfection, (1×105) cells per well were plated and incubated overnight. A mixture of typically 200 ng of Gal4 responsive luciferase reporter plasmid, 10 ng of β-actin-β-galactosidase internal control, 10 ng of GAL-AR LBD or empty vector control, and 10-100 ng of β-catenin or empty vector control were mixed with 0.5 μL of transfection reagent from BioRad and incubated for 20 min and then plated in 24 well plate triplicates. Cells were induced with 1 nM DHT and 0.1 nM-30 uM test compounds after 3 hr and then incubated over night. Cells were collected, and pellets were lysed in 100 μL of 100 mM Tris-HCl (pH 7.5) containing 0.1% Triton X-100. Luciferase and β-galactosidase activities were measured using the Luciferase Assay System (Promega) and Galacto-Light Plus-galactosidase reporter gene assay system (Applied Biosystems), according to the manufacturer's instructions.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 431.37, ALogP 2.27, HBA 6, HBD 2, PSA 120.15, QED 0.55
Compound (SMILES): CCc1c(-c2ccc(OC)c(OC)c2)[nH]c2ccc(-c3noc(CCN(C)C)n3)cc12
InChIKey: JOSRTWNMZFPXHE-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assays: 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-κc Band 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 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-Bue 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 = 11281 nM (pChEMBL 4.949999809265137)
Descriptors: MW 420.51, ALogP 4.57, HBA 6, HBD 1, PSA 76.41, QED 0.45
Compound (SMILES): Cc1nc(C(=O)N2CCC[C@@H](C)[C@H]2CNc2ccc(C(F)(F)F)cn2)c(-c2ccc(F)cc2)s1
InChIKey: SVCKMOGUOXOQCC-AUUYWEPGSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Cell-based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHO-OX1 or CHO-OX2 cells are seeded into black-walled clear-base 384-well plates (Costar) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and selection antibiotic (500 ug/ml G418 or 15 ug/ml Blasticidin) and cultured overnight. The cells are incubated with equal volume of calcium4 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 30 min, followed by putative OX1 or OX2 receptor antagonists (dose-range 0.1 nM-10 μM) for another 30 min.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 492.54, ALogP 6.02, HBA 5, HBD 1, PSA 58.12, QED 0.44
Compound (SMILES): COc1ccc2c(c1)[C@]1(C[C@H]1c1ccc3c(-c4ccc(CCN5CCOCC5)cc4)n[nH]c3c1)C(=O)N2
InChIKey: QZWPJRYZOFVBGD-QCDSWUKFSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Inhibition Assay: Aurora A inhibition was determined using the Z-Lyte assay kit from Invitrogen. The assay was performed using the recommended manufacturer's instructions with 20 μM ATP and 12 nM Aurora A (Invitrogen cat #PV3612). The % inhibition values were determined according to the manufacturer's directions and IC50 values were obtained using a non-linear 4 point logistic curve fit (XLfit4, IDBS)
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 494.6, ALogP 4.49, HBA 5, HBD 2, PSA 79.48, QED 0.41
Compound (SMILES): CN1CCN([C@H]2CC[C@@H](CS(=O)(=O)NNC(=O)c3cc(-c4cn(C)cn4)cc(Cl)c3F)CC2)C1=O
InChIKey: HSDXQVRUJGFEGV-FZNQNYSPSA-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 527.02, ALogP 2.37, HBA 6, HBD 2, PSA 116.64, QED 0.54
Compound (SMILES): CNS(=O)(=O)CC1CCC(n2cnc3cnc4[nH]ccc4c32)CC1
InChIKey: IOJYFDLNTJHKIW-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Caliper mobility shift assay: All four kinases of the JAK/TYK-kinase family were used as purified recombinant GST-fusion proteins, containing the active kinase domains. GST-JAK1(866-1154), GST-JAK3(811-1124), and GST-TYK2(888-1187) were expressed and purified by affinity chromatography at the EPK biology unit.The kinase assays were based on the Caliper mobility shift assay using the LabChip 3000 systems. This technology is similar to capillary electrophoresis and uses charge driven separation of substrate and product in a microfluidic chip.All kinase reactions were performed in 384 well microtiter plates in a total reaction volume of18 μI. The assay plates were prepared with 0.1 μl per well of test compound in the appropriate test concentration, as described under the section 'preparation of compound dilutions'. The reactions were started by combining 9 μl of substrate mix (consisting of peptide and ATP) with 9 μl of kinase dilution. The reactions were incubated for 60 minutes at 30° C. and stopped by adding 70 μl of stop buffer (100 mM Hepes, 5% DMSO, 0.1% Coating reagent, 10 mM EDTA, 0.015% Brij 35).
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 347.44, ALogP 2.19, HBA 5, HBD 2, PSA 92.67, QED 0.76
Compound (SMILES): O=C(Nc1nc2cccc(-c3ccc(C(=O)N4CCCCC4)cc3)n2n1)C1CC1
InChIKey: OADOXVJZNUMIML-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK3 Inhibition Assay: Recombinant human JAK3 catalytic domain (amino acids 781-1124; catalog number PV3855) was purchased from Invitrogen. 0.025 mU of JAK3 was incubated with 2.5 μg polyGT substrate (Sigma catalog number P0275) in kinase reaction buffer (25 mM Tris pH 7.5, 0.5 mM EGTA, 0.5 mM Na3VO4, 5 mM b-glycerophosphate, 0.01% Triton X-100, 1 μM non-radioactive ATP, 0.25 μCi 33P-gamma-ATP (GE Healthcare, catalog number AH9968) final concentrations) with or without 5 μL containing test compound or vehicle (DMSO, 1% final concentration), in a total volume of 25 μL, in a polypropylene 96-well plate (Greiner, V-bottom). After 105 min at 30° C., reactions were stopped by adding of 25 μL/well of 150 mM phosphoric acid. All of the terminated kinase reaction was transferred to prewashed (75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). Plates were washed 6 times with 300 μL per well of a 75 mM phosphoric acid solution and the bottom of the plates was sealed. 40 μL/well of Microscint-20 was added, the top of the plates was sealed and readout was performed using the Topcount (Perkin Elmer). Kinase activity was calculated by subtracting counts per minute (cpm) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as:Percentage inhibition=((cpm determined for sample with test compound present−cpm determined for sample with positive control inhibitor) divided by (cpm determined in the presence of vehicle−cpm determined for sample with positive control inhibitor))*100%.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 389.46, ALogP 3.37, HBA 5, HBD 1, PSA 79.6, QED 0.74
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CCN(CCNS(C)(=O)=O)CC4)cc3c2C(C)C)cn2ncnc2c1C
InChIKey: RMRGHHVCYMMIPQ-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 = 2020 nM (pChEMBL 5.699999809265137)
Descriptors: MW 508.69, ALogP 4.35, HBA 6, HBD 2, PSA 95.39, QED 0.39
Compound (SMILES): CCOc1cc(-c2ccc(N3CC[C@H](NC(=O)OC(C)(C)C)[C@@H](OCCN(C)C)C3)nc2)c2c(C#N)cnn2c1
InChIKey: BOVOHHVSVDWCBO-DQEYMECFSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET G810R Mutant Assay: The potency of a compound inhibiting G81 OR mutant RET kinase was determined using CisBio's HTRF Kinease-TK assay technology. The assays contained G81OR mutant RET produced at Array Biopharma, Inc. (1 nM enzyme p1982 Lot. No. 160713. The kinase was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog #62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared as a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-min incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-Ab-Cryptate in HTRF detection buffer (all from CisBio, part of cat #62TK0PEC). After a 1-h incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. One hundred POC was determined using no test compounds, and 0 POC was determined using pre-quenched control reactions. A 4-parameter logistic curve was fit to the POC values as a function of the concentration of compound, and the IC50 value was the point where the best-fit curve crossed 50 POC.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 549.68, ALogP 3.72, HBA 10, HBD 1, PSA 117.25, QED 0.43
Compound (SMILES): O=C(NC1CC(F)(F)C1)[C@H](c1ccccc1Cl)N(C(=O)[C@@H]1CCC(=O)N1c1cccc(Cl)n1)c1cccc(F)c1
InChIKey: KGVKTOYOZORCGW-OFVILXPXSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: In Vitro Assay: A test compound is prepared as 10 mM stock in DMSO and diluted to 50× final concentration in DMSO, for a 50 μl reaction mixture. IDH enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutaric acid is measured using a NADPH depletion assay. In the assay the remaining cofactor is measured at the end of the reaction with the addition of a catalytic excess of diaphorase and resazurin, to generate a fluorescent signal in proportion to the amount of NADPH remaining. IDH1-R132 homodimer enzyme is diluted to 0.125 μg/ml in 40 μl of Assay Buffer (150 mM NaCl, 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 0.05% BSA, 2 mM b-mercaptoethanol); 1 μl of test compound dilution in DMSO is added and the mixture is incubated for 60 minutes at room temperature. The reaction is started with the addition of 10 μl of Substrate Mix (20 μl NADPH, 5 mM alpha-ketoglutarate, in Assay Buffer) and the mixture is incubated for 90 minutes at room temperature. The reaction is terminated with the addition of 25 μl of Detection Buffer (36 μg/ml diaphorase, 30 mM resazurin, in 1× Assay Buffer), and is incubated for 1 minute before reading on a SpectraMax platereader at Ex544/Em590.Compounds are assayed for their activity against IDH1 R132C following the same assay as above with the following modifications: Assay Buffer is (50 mM potassium phosphate, pH 6.5; 40 mM sodium carbonate, 5 mM MgCl2, 10% glycerol, 2 mM b-mercaptoethanol, and 0.03% BSA). The concentration of NADPH and alpha-ketoglutarate in the Substrate Buffer is 20 μM and 1 mM, respectively.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 591.42, ALogP 5.71, HBA 4, HBD 1, PSA 82.61, QED 0.36
Compound (SMILES): CCC(NC(=O)O[C@@H]1CC[C@H](c2cc(NC(=O)Cc3ccc(C)nc3)n[nH]2)C1)C(F)(F)F
InChIKey: ZBKYUHYCKHIUOK-BTPDTDQASA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: Biochemical Assay: The purpose of CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 full length catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2, CPC Scientific, Sunnyvale, Calif.) (SEQ ID NO:1). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Wild-type CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Matl enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA.
Measurement: Ki = 0.47 nM (pChEMBL 9.329999923706055)
Descriptors: MW 453.47, ALogP 4, HBA 5, HBD 3, PSA 109, QED 0.59
Compound (SMILES): O=C1CCCN1CC1(O)CCN(c2nc(Oc3cccc(F)c3)cc(C(F)(F)F)n2)CC1
InChIKey: PHPIBQGNEKZAKD-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Competition Binding Assay: The affinity of the compounds of the invention to the human DAT or NET or SERT transporters is assessed by using the [3H]WIN-35,428 or [3H]nisoxetine or [3H]citalopram binding assays in recombinant human DAT, NET and SERT membranes with the SPA technology. The final assay volume is 50 μL in 384 well plates.Briefly, 0.5 μL of test compound in neat DMSO or 0.5 μL of DMSO for total binding (TB) or 0.5 μL of indatraline 1 mM (10 μM final concentration) for non specific binding (NSB) are added to the assay plate. 50 μL of the SPA mixture is added to each well, containing: 30 μg/mL or 10 μg/mL or 25 μg/mL DAT, NET, SERT membranes, respectively; 5 nM [3H]WIN-35,428 or 5 nM [3H]nisoxetine or 1 nM [3H]citalopram, for DAT, NET, SERT assay, respectively; 2.5 mg/mL or 1 mg/mL or 4 mg/mL WGA-PVT SPA beads (PerkinElmer RPNQ0001, for DAT, NET, SERT assay, respectively. All components are added to Assay Buffer (20 mM HEPES pH 7.4, 145 mM NaCl, 5 mM KCl, 0.01% Pluronic F-127). 0.02% BSA was used for DAT binding only. Plates are sealed with Topseal A and centrifuged 1 min, 800 rpm. Plates are loaded into a 1450 Microbeta TriLux (Perkin-Elmer) plate reader and the radioactivity counted after at least 4 hrs or overnight incubation at room temperature. Curve fitting and IC50 estimations are performed using a four parameter model in XLfit (IDBS, Guilford, UK) for Microdoft Excel (Microsoft, Redmond, Wash.).
Measurement: IC50 < 100000 nM (pChEMBL 4)
Descriptors: MW 454.42, ALogP 3.38, HBA 6, HBD 1, PSA 78.79, QED 0.7
Compound (SMILES): C[C@H](F)CN1CCC(C(=O)Nc2cc3cc(-c4cnn(C)c4)ccc3cn2)CC1
InChIKey: QRLRNSOZYZXEOE-HNNXBMFYSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: The screening assay for GSK3beta kinase activity: Each compound is dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 11-point dose-response curves from 10 μM to 0.0003 M) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 1536-well black-walled round bottom plates (Corning).The GSK3β kinase assay is run using the Ser/Thr 09 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologiesa Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as ratio of coumarin emission/fluorescein emission.Briefly, recombinant GSK3β kinase, ATP and Ser/Thr peptide 09 are prepared in 1× Kinase buffer to final concentrations of 0.04 μg/mL, 46 μM, and 4 μM respectively. The mixture is allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 serve as control reactions.After incubation, diluted Development Buffer is added to the reaction and allowed to further incubate for one hour at room temperature. The plate is read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer).The Emission ratio (Em) is calculated as a ratio of the coumarin (C) emission signal (at 445 nm)/Fluorescein (F) emission signal (at 520 nm). The percent phosphorylation is then calculated using the following formula: [1−((Em ratio×F100%)−C100%)/((C0%−C100%)+(Em ratio×(F100%−F0%)))].Dose-response curves are generated and inhibitory concentration (IC50) values are calculated using non-linear regression curve fit in the Dotmatics' Studies Software (Bishops Stortford, UK).
Measurement: EC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 395.48, ALogP 3.64, HBA 5, HBD 1, PSA 63.05, QED 0.72
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CCC(NC5(CO)COC5)CC4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: RAZVTRHTHJSSNE-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: 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 = 0.94 nM (pChEMBL 9.029999732971191)
Descriptors: MW 488.64, ALogP 4.39, HBA 7, HBD 3, PSA 100.36, QED 0.37
Compound (SMILES): O=C(OC(C(F)(F)F)C(F)(F)F)N1CCCc2ccccc2C1
InChIKey: LBAQRUZWGHHEQO-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: Fluorescent Assay: Rat/homo FAAH:Procedure was followed as described for hMGL, except that arachidonoyl-methyl coumarin (was used as fluorigenic substrate. Compounds were diluted in 50:50 DMSO/assay buffer (50 mM HEPES, 1 mM EDTA, 0.1% BSA, pH 7.4) so as to have a final DMSO concentration below 8% in each reaction. For the screening assay, 3 concentrations (1 μM, 10 μM, and 100 μM) of test compounds, 15 jag of ΔTM rFAAH or hRAAH and assay buffer were pre-incubated for 15 min at 25° C. AAMCA (20 μM, 2×Km) was added prior to incubation at 25° C. and kinetic fluorescence reading every 20 minutes (λex=360/λem=460) for 4 hours on a BioTek Synergy HT Microplate Reader.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 341.25, ALogP 4.06, HBA 2, HBD 0, PSA 29.54, QED 0.72