lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nc[nH]c1CN1CCC2(CC1)COC1c3cccc(Cl)c3OC(C)(C)C1C2
InChIKey: QNOXPCLUMHQZFP-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Test for Evaluating hERG Inhibition: To a cultured CHO cell strain which stably expresses hERG was added each test compound to adjust the final concentration of DMSO to 0.0135 to 0.5%. The hERG current was measured with QPatch HT (Sophion Inc.), and the concentration at which 50% of the hERG current was inhibited by each test compound (IC50 value; μM) was calculated.
Measurement: IC50 = 400 nM (pChEMBL 6.400000095367432)
Descriptors: MW 415.97, ALogP 4.9, HBA 4, HBD 1, PSA 50.38, QED 0.76
Compound (SMILES): CC(=O)c1cn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)Nc2cccc(Br)n2)c(C)n1
InChIKey: QTVFLCRZSVVQFW-OCCSQVGLSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 min using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of Complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 452.28, ALogP 2.13, HBA 6, HBD 1, PSA 97.19, QED 0.55
Compound (SMILES): CCNC(=O)c1cn2c(NCC)nc(-c3ccccc3)c(-c3cc(C)nc(C)c3)c2n1
InChIKey: NGVCANAVHADHLZ-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: A2A Tag-lite HTRF Assay: Assays were conducted in black low volume 384-well polystyrene plates (Greiner 784076-25) in a final volume of 10 μL. Test compounds were first serially diluted in DMSO and 100 nl added to the plate wells before the addition of other reaction components. The final concentration of DMSO was 1%. Tag-lite® Adenosine A2A labeled cells (CisBio C1TT1A2A) were diluted 1:5 into Tag-lite buffer (CisBio LABMED) and spun 1200 g for 5 mins. The pellet was resuspended at a volume 10.4× the initial cell suspension volume in Tag-lite buffer, and Adenosine A2A Receptor Red antagonist fluorescent ligand (CisBio L0058RED) added at 12.5 nM final concentration. 10 ul of the cell and ligand mix was added to the assay wells and incubated at room temperature for 45 minutes before reading on a PHERAstar FS plate reader (BMG Labtech) with HTRF 337/620/665 optical module. Percent binding of the fluorescent ligand was calculated; where 100 nM of A2A antagonist control ZM 241385 (Tocris 1036) displaces the ligand 100% and 1% DMSO has 0% displacement. The % binding data versus the log of the inhibitor concentration was fitted to a one-site competitive binding model (GraphPad Prism version 7.02) where the ligand constant=12.5 nM and the ligand Kd=1.85 nM.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 414.51, ALogP 4.26, HBA 6, HBD 2, PSA 84.21, QED 0.49
Compound (SMILES): Cc1nc(-c2ccc(Sc3ccc(Cl)cc3)c(C#N)c2)[se]c1C(=O)O
InChIKey: PRZPDOMUBWBXLH-UHFFFAOYSA-N
Target: Xanthine dehydrogenase/oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P47989
Assay: Inhibition Assay: The inhibition of the activity of xanthine oxidase (XO) is tested through a coupled enzymatic reaction of xanthine oxidase, horseradish peroxidase (HRP) and a substrate thereof. First, xanthine oxidase oxidizes hypoxanthine to produce xanthine and hydrogen peroxide, and further oxidizes xanthine to produce uric acid and hydrogen peroxide. Then, hydrogen peroxide reacts with 10-acetyl-3,7-dihydroxyphenoxazine (Ampliflu Red) under catalytic action of horseradish peroxidase so as to produce resorufin, a compound with strong fluorescence. The fluorescence intensity of resorufin is determined by using a fluorescence microplate, which is in direct proportion to the activity of xanthine oxidase.
Measurement: IC50 = 3.58 nM (pChEMBL 8.449999809265137)
Descriptors: MW 433.78
Compound (SMILES): Cc1cc(C2CCCN(Cc3ccc(C4CC4)cc3)C2)n2ncnc2n1
InChIKey: UARWRGCFJGZDPT-UHFFFAOYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Enzymatic Assay: An IMAP TR-FRET-based phosphodiesterase assay was developed using the PDE2A isoform. IMAP technology is based on high-affinity binding of phosphate by immobilized metal (MIII) coordination complexes on nanoparticles. The IMAP binding reagent recognizes phosphate groups on AMP or GMP generated from cAMP or cGMP in a PDE reaction. Cyclic nucleotides that carry a phosphodiester bond and not a free phosphate are not recognized by the binding reagent. The time resolved fluorescence resonance energy transfer (TR-FRET) is afforded by a Terbium (Tb)-Donor pre-bound to the nanoparticles. FRET occurs when the fluorescent-labeled AMP or GMP product of a PDE reaction binds and comes into close proximity to the Tb-Donor complex. Due to the long lifetime of Tb fluorescence, detection can be run in time-resolved mode to reduce or eliminate interference from auto-fluorescent compounds.The IMAP TR-FRET PDE2A assay was performed in 1536-well white plates. A total of 250 pg per well of FLAG-tagged PDE2A1 (amino acids 2-941) was dispensed in 2.5 μL IMAP assay buffer consisting of 10 mM Tris pH 7.2, 10 mM MgCl2, 1 mM DTT, and 0.1% fatty acid free BSA. 30 nL of compound was then added from 1 mM stocks in DMSO using a Kalypsys Pintool. Plates were incubated for 5 min at room temperature before dispensing 1.5 μL of 533 nM FAM-cAMP substrate for a final concentration of 200 nM. Following a brief centrifugation, plates were incubated for 30 min at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent Tb complex to each well which was prepared according to manufacturer's recommendations (Molecular Devices). Plates were incubated an additional 90 minutes at room temperature and read on a Viewlux plate reader. All compounds were solvated at a concentration of 10 mM in DMSO and tested in 11-point half-log dose-response. Curve fitting and IC50 values were determined using a standard four parameter fit.PDE2
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 347.47, ALogP 3.69, HBA 5, HBD 0, PSA 46.32, QED 0.72
Compound (SMILES): COCCOCCOCCOCCOCCNc1nc2ccc3c(c2s1)[C@@]12CCCC[C@H]1[C@@H](C3)N(C)CC2
InChIKey: JWEMIEDKYWEOKW-ZLZBJJAKSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Receptor Binding Assay: Affinities of various compounds were measured in vitro using competitive radioligand binding assays. Serial dilutions of test compounds were incubated with membranes prepared from CHO-K1 cells expressing the mu opioid receptor (MOR; for opioid receptor binding) or rat forebrain membranes (for NMDA receptor binding). 2 nM [3H] Naloxone (MOR) or 0.2 nM [3H] MK801 (NMDA) were used as the specific, competitive radioligands. 10 uM Naloxone (MOR), or 10 uM MK801 (NMDA) was used to determine non-specific binding. Bound radioactivity was measured using a scintillation counter & IC50 values for test compounds were determined by non-linear regression analysis using a one-site competition model (Graph Pad Prizm). Due to each test compound being solubilized in a buffered system for the assay, the results presented herein reflect the free base activity.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 547.76, ALogP 4.11, HBA 9, HBD 1, PSA 74.31, QED 0.31
Compound (SMILES): CCC(C)N1CCc2nn(-c3c(C)cccc3OCC(C)C)c(-c3ccc(NC(N)=O)c(F)c3)c2C1
InChIKey: FFAKJGWWSBIACN-UHFFFAOYSA-N
Target: C5a anaphylatoxin chemotactic receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21730
Assay: chemotaxis assay: Optionally, compounds may be further assayed for their ability to inhibit chemotaxis in cells. Chemotaxis assays are performed using 5 μm pore polycarbonate, polyvinylpyrrolidone-coated filters in 96-well chemotaxis chambers (Neuroprobe; Gaithersburg, Md.) using chemotaxis buffer (Hank's balanced salt solution (HBSS) and 1% FBS). C5aR ligands (i.e., C5a, R&D Systems; Minneapolis, Minn.) are use to evaluate compound mediated inhibition of C5aR mediated migration. Other chemokines (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) are used as specificity controls. The lower chamber is loaded with 29 μl of chemokine (i.e., 0.03 nM C5a) and varying amounts of compound; the top chamber contains 100,000 U937 or neutrophil cells in 20 μl. The chambers are incubated 1.5 hours at 37° C., and the number of cells in the lower chamber quantified either by direct cell counts in five high powered fields per well or by the CyQuant assay (Molecular Probes), a fluorescent dye method that measures nucleic acid content and microscopic observation.
Measurement: IC50 = 27.5 nM (pChEMBL 7.559999942779541)
Descriptors: MW 493.63, ALogP 5.67, HBA 5, HBD 2, PSA 85.41, QED 0.42
Compound (SMILES): Cc1nc(N2CCOCC2)ccc1Nc1nc2ccc(-c3ccc(NC(=O)Cc4ccc(F)cc4)cc3)cn2n1
InChIKey: UQFTWYMNTXMHEH-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Mps-1 Kinase Assay: For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of Mps-1 in assay buffer [0.1 mM sodium-ortho-vanadate, 10 mM MgCl2, 2 mM DTT, 25 mM Hepes pH 7.7, 0.05% BSA, 0.001% Pluronic F-127] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to Mps-1 before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μl of a solution of 16.7 adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μl assay volume is 10 μM) and peptide substrate (1.67 μM=>final conc. in the 5 μl assay volume is 1 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of Mps-1 in the assay was adjusted to the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical enzyme concentrations were in the range of about 1 nM (final conc. in the 5 μl assay volume). The reaction was stopped by the addition of 3 μl of a solution of HTRF detection reagents (100 mM Hepes pH 7.4, 0.1% BSA, 40 mM EDTA, 140 nM Streptavidin-XLent [#61GSTXLB, Fa. Cis Biointernational, Marcoule, France], 1.5 nM anti-phospho(Ser/Thr)-Europium-antibody [#AD0180, PerkinElmer LAS, Rodgau-Jügesheim, Germany].The resulting mixture was incubated 1 h at 22° C. to allow the binding of the phosphorylated peptide to the anti-phospho(Ser/Thr)-Europium-antibody. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Europium-labelled anti-phospho(Ser/Thr) antibody to the Streptavidin-XLent. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured in a Viewlux TR-FRET reader (PerkinElmer LAS, Rodgau-Jügesheim, Germany). The “blank-corrected normalized ratio” (a Viewlux specific readout, similar to the traditional ratio of the emissions at 665 nm and at 622 nm, in which blank and Eu-donor crosstalk are subtracted from the 665 nm signal before the ratio is calculated) was taken as the measure for the amount of phosphorylated substrate. The data were normalised (enzyme reaction without inhibitor=0% inhibition, all other assay components but no enzyme=100% inhibition).
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 537.6, ALogP 5, HBA 8, HBD 2, PSA 96.68, QED 0.3
Compound (SMILES): CC1(C)Cc2c(C#N)c(N3CCN(C(=O)c4ccco4)CC3)nc(C3CCCCC3)c2CO1
InChIKey: QBQVOHKKAZQTGK-UHFFFAOYSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Inhibition Assay: Assays were conducted in a volume of 76 μl assay buffer (150 mM NaCl, 10 mM MgCl2, 20 mM Tris pH 7.5, 0.03% bovine serum albumin) as follows in a standard 384-well plate: To 25 ul of substrate mix (8 uM NADPH, 2 mM aKG), 1 μl of test compound was added in DMSO. The plate was centrifuged briefly, and then 25 μl of enzyme mix was added (0.2 μg/ml IDH1 R132H) followed by a brief centrifugation and shake at 100 RPM. The reaction was incubated for 50 minutes at room temperature, then 25 μl of detection mix (30 μM resazurin, 36 μg/ml) was added and the mixture further incubated for 5 minutes at room temperature. The conversion of resazurin to resorufin was detected by fluorescent spectroscopy at Ex544 Em590 c/o 590.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 448.57, ALogP 4.41, HBA 6, HBD 0, PSA 82.6, QED 0.69
Compound (SMILES): CCOC(=O)NCCN1CCN(c2ccc3nc(CC)c(N(C)c4nc(-c5ccc(F)cc5)cs4)n3n2)CC1
InChIKey: YJVRDEJFQPEUBC-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Biochemical Assay: Compound IC50 values are determined in a fluorescent hENPP2 (UniProtKB/SwissProt Sequence ref Q13822) biochemical assay using the fluorogenic autotaxin substrate FS-3 as substrate. FS-3 is a doubly labeled analog of LPC wherein the fluorophore is quenched through intramolecular energy transfer. Without hENPP2, the emission of the probe is quenched. If the substrate is hydrolyzed by hENPP2, the emission of the probe is not quenched anymore resulting in a fluorescence increase. Inhibition of hENPP2 by compounds will result in a decrease of the signal. 10 uL of a dilution series of compound, starting from 20 uM highest concentration, dilution, is added to the wells. hENPP2 is used at a final concentration of 0.4 ug/mL or 0.64 ug/mL (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration). The enzyme is diluted in 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% fatty acid free BSA in a total volume of 20 uL. Enzyme mixture is added to compounds and the resulting mixture is incubated for 30 min at room temperature under shaking. The reaction is started by the addition of 20 uL of 0.75 uM FS-3 diluted in the same buffer as described above and the mixture is incubated at 30° C. for 30 min. Fluorescence is read on the Envision (Excitation 485 nm, emission 520 nM).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 552.68, ALogP 4.19, HBA 10, HBD 1, PSA 91.13, QED 0.33
Compound (SMILES): Cn1ncc(NC(=O)c2csc(-c3n[nH]c4c3CCC4)n2)c1[C@@H]1CC[C@@H](N)[C@H](F)CO1
InChIKey: JRVNRDSSUXSRDJ-HSMVNMDESA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Biological Assay: Determination of the Pim kinase activity of a Formula I compound is possible by a number of direct and indirect detection methods. Certain exemplary compounds described herein were assayed for their Pim kinase binding activity, including isoforms Pim-1, Pim-2, andPim-3, (Example 901) and in vitro activity against tumor cells (Example 902). Certain exemplary compounds of the invention had Pim binding activity IC50 values less than about 1 micromolar (μM). Certain compounds of the invention had tumor cell-based activity EC50 values less than about 1 micromolar (μM).
Measurement: Ki = 0.03 nM (pChEMBL 10.520000457763672)
Descriptors: MW 445.52, ALogP 2.52, HBA 8, HBD 3, PSA 123.74, QED 0.57
Compound (SMILES): Cc1cc(-c2ncnc(Nc3ccn(C)n3)n2)ccc1CNC(=O)c1noc(C(C)(C)CF)n1
InChIKey: MJIKMXCOKNKQJU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In Vitro Kinase Assay: The purpose of the BTK in vitro assay is to determine compound potency against BTK through the measurement of IC50. Compound inhibition is measured after monitoring the amount of phosphorylation of a fluorescein-labeled polyGAT peptide (Invitrogen PV3611) in the presence of active BTK enzyme (Upstate 14-552), ATP, and inhibitor. The BTK kinase reaction was done in a black 96 well plate (costar 3694). For a typical assay, a 24 pL aliquot of a ATP/peptide master mix (final concentration; ATP 10 kM, polyGAT 100 nM) in kinase buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 200 μM Na3PO4, 5 mM DTT, 0.01% Triton X-100, and 0.2 mg/ml casein) is added to each well. Next, I pL of a 4-fold, 40× compound titration in 100% DMSO solvent is added, followed by adding 15 μL of BTK enzyme mix in 1× kinase buffer (with a final concentration of 0.25 nM). The assay is incubated for 30 minutes before being stopped with 28 μL of a 50 mM EDTA solution. Aliquots (5 μL) of the kinase reaction are transferred to a low volume white 384 well plate (Corning 3674), and 5 μL of a 2× detection buffer (Invitrogen PV3574, with 4 nM Tb-PY20 antibody, Invitrogen PV3552) is added. The plate is covered and incubated for 45 minutes at room temperature. Time resolved fluorescence (TRF) on Molecular Devices M5 (332 nm excitation; 488 nm emission; 518 nm fluorescein emission) is measured. IC50 values are calculated using a four parameter fit with 100% enzyme activity determined from the DMSO control and 0% activity from the EDTA control.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 465.49, ALogP 2.88, HBA 10, HBD 2, PSA 136.54, QED 0.4
Compound (SMILES): CC(C)(C)c1noc(C23CCC(CN(C(=O)C45CC(F)(C4)C5)c4cccc(-c5ccc(NS(C)(=O)=O)nc5)c4)(CC2)CC3)n1
InChIKey: ZIRMQLDHLAEBBL-UHFFFAOYSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: transient human FXR/Gal4-luciferase reporter assay: A Gal4-hFXR fusion construct reporter system was used as the primary assay to characterize compound activity. A construct including 5 copies of the Gal4 promoter response element upstream of a firefly luciferase reporter cDNA was stably expressed in HEK293 cells. This reporter cell line was maintained in Dulbecco's Modified Eagle's medium (DMEM; Gibco) supplemented with 1% penicillin-streptomycin (P/S) solution, 500 μg/mL Zeocin and 10% charcoal/dextran-treated fetal bovine serum (cs-FBS) at 37° C. in a humidified 5% CO2 atmosphere. Another plasmid was constructed in which the human cytomegalovirus promoter in the pcDNA3.1 vector directs the expression of the cDNA encoding a fusion protein comprised of the DNA binding domain from the Gal4 transcription factor fused to the ligand binding domain from human FXR.The day prior to transfection, the reporter cells in culture are detached from the plate with trypsin and plated into a T75 flask at a sufficient density to achieve approximately 90% confluence the next morning. The transfection reagents are prepared by separately diluting 25 μg of the pcDNA3.1-Gal4-FXR plasmid into 1.87 mL of Opti-MEM (Thermo-Fisher), and 40 μL of Lipofectamine 2000 (Thermo-Fisher) into 1.87 mL of Opti-MEM, and then adding the diluted DNA solution into the diluted Lipofectamine 2000 solution and incubating at room temperature for 15-20 minutes. The mixture is further diluted with 10 mL of a solution comprised of DMEM, 10% cs-FBS, and 1% P/S immediately prior to transferring to the cells. The maintenance culture media is aspirated from the cells and the final transfection mixture is added before the cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. This protocol can be scaled up, and the transiently transfected cells can be cryopreserved in an assay-ready format.For compound testing, 100 nL of the compounds (serial dilutions in DMSO) are dispensed with an Echo acoustic dispenser (Labcyte) into the wells of a Corning/Costar clear bottom 384-well white plate. The transfected cells are harvested, counted, and diluted such that 10-25,000 cells in 25 μL are plated into each well of the 384-well compound assay plate. The compound-treated cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. The next morning 25 μL of Steady-Glo (Promega) are added to each well of the plate, the mixture is incubated for 15 min. with shaking, and luminescence is measured on an Envision (Perkin Elmer) plate reader. Background counts from cells treated with DMSO alone are subtracted from all raw counts, and the corrected values are converted to a percentage of the control response attained with 8 μM GW-4064. These data are fit to a 4-parameter log agonist-response equation to calculate an EC50 value.
Measurement: EC50 = 509 nM (pChEMBL 6.289999961853027)
Descriptors: MW 621.78, ALogP 6.32, HBA 7, HBD 1, PSA 118.29, QED 0.31
Compound (SMILES): CC(=O)N1CCc2c(c(Nc3cc(F)c(-c4cn(C)nc4C(F)(F)F)cc3F)nn2C2COC2)C1
InChIKey: IVFSNNBSBKKKDU-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (6×His disclosed as SEO ID NO: 3) (Perkin Elmer AD0110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 496.44, ALogP 3.8, HBA 7, HBD 1, PSA 77.21, QED 0.56
Compound (SMILES): N#Cc1c(N2CCC3(CCC3)CC2)c2cc(C3CC3)ncc2[nH]c1=O
InChIKey: RAJAKUIIXAWIFY-UHFFFAOYSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Enzymology Assay: 1. Experimental materials and instrumentsPDE9A2 Enzyme (BPS, Cat. No. 60090)384-well plate (Perkin Elmer, Cat. No. 6007279)2. Experimental procedurePreparation of the compounds: the compounds were prepared into 10 mM compound stock solution in DMSO for long-term storage. The obtained compound stock solution was diluted in 100 times with DMSO to obtain 100 μM compound working mother solution, and then the compound working mother solution was diluted in 3 times with DMSO to obtain 8-10 concentration gradients of diluted compound mother liquor (100×).Incubation with the compounds: A very small amount of liquid pipetting system Echo was used to pipette the diluted compound mother liquor into a 384-well plate. 200 nL diluted compound mother liquor and 10 μL PDE9A2 enzyme solution were added to each compound well. After centrifugation at 1000 rpm for 1 min, the mixture was incubated for 15 min at the room temperature. Then the 10 μL substrate mixture was added. After centrifugation at 1000 rpm for 1 min, the mixture was incubated with shocking for 30 min at the room temperature. Finally, a stop solution was added to end the reaction system. The mixture was incubated with shocking for 60 min at the room temperature. In the maximum reading hole (Max), the compound was replaced by solvent. In the minimum reading hole (Min), the compound and enzyme solution were replaced by solvent.
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 334.42, ALogP 3.44, HBA 4, HBD 1, PSA 72.78, QED 0.91
Compound (SMILES): CC(=O)c1nn(CC(=O)N2C[C@@]3(F)CNC(=O)C(C)(C)CCCCOCc4ccc(Br)nc4NC(=O)[C@@H]2C3)c2cnc(-c3cnc(C)nc3)cc12
InChIKey: MAIHWNOXCNPYGI-CYFIQEHSSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 M each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 minutes using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of Complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 764.66, ALogP 4.35, HBA 11, HBD 2, PSA 174.19, QED 0.22
Compound (SMILES): CN(C(=O)C1CCOCC1)c1c[nH]/c(=N\c2ccc(Oc3ccon3)cc2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: ADRWFGBOPFLVAG-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 3000 cells/well and cultured in the medium (7.0% fetal bovine serum, 7.0% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 1.37 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH7.5), and each well was added with 20 μL of this buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 384 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 535.99, ALogP 4.28, HBA 8, HBD 1, PSA 114.95, QED 0.38
Compound (SMILES): CC1(c2ccccc2)NC(=O)C2(CCN(C#N)C2)NC1=O
InChIKey: KKTNBBTYQULHLY-UHFFFAOYSA-N
Target: OTU domain-containing protein 7B (SINGLE PROTEIN), Homo sapiens
UniProt: Q6GQQ9
Assay: Biochemical Assay: Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series to be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl of 50% DMSO or diluted compound was added to the plate. Cezanne 1 was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mM-beta-mercaptoethanol) to the equivalent of 0.005 μl/well and 10 μl of diluted Cezanne 1 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50 nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2 hr incubation at room temperature. Readings were performed on a Pherastar Plus. λ Excitation 540 nm; λ Emission 590 nm.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 284.32, ALogP 0.07, HBA 4, HBD 2, PSA 85.23, QED 0.71
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@@H](C)Cc2ncc(F)cn2)nnc1-c1cc(C)ccn1
InChIKey: UJPLEGRDPQKCQE-HNNXBMFYSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 M GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 M GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.) to obtain EC50.
Measurement: EC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 513.56, ALogP 2.96, HBA 10, HBD 1, PSA 134.01, QED 0.36
Compound (SMILES): O=C(NCC(F)(F)F)Nc1cccc(-c2cnc3cc(-c4cnn(C5CCN(C(=O)C6(O)CC6)CC5)c4)cnn23)c1
InChIKey: ACFZIIYFPBHHFC-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: FGFR3 Enzymatic Assay: The inhibitor potency of compounds of the invention was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. A 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated peptide and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/ml BSA, pH 7.8; added fresh 30 mM EDTA and Perkin Elmer Lance Reagents for HTRF at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader.GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 568.56, ALogP 3.63, HBA 8, HBD 3, PSA 129.68, QED 0.33
Compound (SMILES): O=C(c1cnn[nH]1)N1CC2CCC(C1)N2S(=O)(=O)c1cccc(Cl)c1Cl
InChIKey: QVDJTIQGSTVKRO-UHFFFAOYSA-N
Target: Aldo-keto reductase family 1 member C3 (SINGLE PROTEIN), Homo sapiens
UniProt: P42330
Assay: AKR1C3-Inhibitory Activity Assay: The enzyme used was recombinant human AKR1C3 (Aldo-keto reductase family 1 member C3; GenBank Accession No. NM_003739). This was expressed in E. coli as GST (glutathione S transferase) fusion protein and purified by glutathione Sepharose affinity chromatography. The GST was removed by digestion with thrombin and subsequent size exclusion chromatography (Dufort, I., Rheault, P., Huang, X F., Soucy, P., and Luu-The, V., Endocrinology 140, 568-574 (1999)).For the assay, 50 nl of a 100-fold concentrated solution of the test substance in DMSO were pipetted into a black low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2.5 μl of a solution of AKR1C3 in assay buffer [50 mM potassium phosphate buffer pH 7, 1 mM DTT, 0.0022% (w/v) Pluronic F-127, 0.01% BSA (w/v) and protease inhibitor cocktail (Complete, EDTA-free Protease Inhibitor Cocktail from Roche)] were added and the mixture was incubated for 15 min to allow pre-binding of the substances to the enzyme prior to the enzyme reaction. The enzyme reaction was then started by addition of 2.5 μl of a solution of NADPH (20 μM→final concentration in 5 μl of assay volume is 10 μM) and Coumberone (0.6 μM→final concentration in 5 μl of assay volume is 0.3 μM) in assay buffer, and the resulting mixture was incubated at 22° C. for the reaction time of typically 90 min. The concentration of the AKR1C3 and the reaction time was adapted to the respective activity of the enzyme preparation and adjusted such that the assay was carried out in the linear range. Typical AKR1C3 concentrations were in the region of 1 nM. The reaction was stopped by addition of 2.5 μl of a stop solution consisting of 3 μM EM-1404 as inhibitor (U.S. Pat. No. 6,541,463) in 50 mM HEPES pH7.5 (3 μM EM-1404→final concentration in 7.5 μl of assay volume is 1 μM). The fluorescence of the Coumberole was then measured at 520 nm (excitation at 380 nm) using a suitable measuring instrument (Pherastar from BMG Labtechnologies). The intensity of the fluorescence was used as a measure of the amount of Coumberole formed and thus of the enzyme activity of AKR1C3. The data were normalized (enzyme reaction without inhibitor=0% inhibition; all other assay components, but no enzyme=100% inhibition). Usually, the test substances were tested on the same microtiter plate at 11 different concentrations in the range from 20 μM to 73 pM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 10.9 nM, 3.1 nM, 0.9 nM, 0.25 nM and 73 pM, the dilution series were prepared prior to the assay on the level of the 100-fold concentrated solution by serial 1:3 dilutions with 100% DMSO) in duplicates for each concentration, and the IC50 values were calculated using a 4-parameter fit.
Measurement: IC50 = 0.8 nM (pChEMBL 9.100000381469727)
Descriptors: MW 416.29, ALogP 1.79, HBA 5, HBD 1, PSA 99.26, QED 0.82
Compound (SMILES): CCOc1nc(C)c2nc(-c3cncc(F)c3)n(Cc3ccccc3OC)c2n1
InChIKey: ZGNALWOGXOWODW-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: Inhibition of the Activation Assay: As human TLR7 expressing cell line, HEK293 cell line was bought from IMGENEX Corporation (TLR7/NF-ηB/SEAPorter HEK293 cell), which is human embryonic kidney cell line and stably expresses full-length human TLR7 gene and secreted alkaline phosphatase (SEAP) reporter gene under the transcriptional control of an NE-ηB response element. The TLR7/NF-ηB/SEAPorter HEK293 cell was cultivated with DMEM containing 10% fetal bovine serum (FBS) and 10 μg/mL blasticidin S at 37° C. in the presence of 5% CO2. The TLR7/NF-ηB/SEAPorter HEK293 cell was seeded into 96-well microtiter plate at 5×104 cell/90 μL/well, and the place was cultivated at 37° C. in a CO2 incubator overnight. Each test compound that was diluted with the medium was added to the wells (10 μL/well), wherein each final concentration was adjusted to 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μmol/L, or 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 μmol/L. After 0.5 hour, R-848 that is TLR7/8 ligand was added to each well (10 μL/well), wherein each final concentration was adjusted to 200 nmol/L. The total volume was adjusted to 110 μL/well, the test samples were incubated in CO2 incubator for 20±1 hours, and then the SEAP activity was measured as activation of TLR7. The SEAP activity was evaluated as follows: p-nitro-phenyl phosphate (pNPP) (Invitrogen) was added to the incubated sample (50 μL/well); after 15 minutes, 4 mol/L sodium hydroxide solution (nacalai tesque) was added thereto (50 μL/well) to quench the reaction; and the absorbance of each sample was measured at 405 nm with microplate reader Elx808 (BioTek). The 50% inhibitory concentration (IC50 value) of each sample compound was calculated based on 100% of the SEAP activity wherein the test sample comprises no sample compound.
Measurement: IC50 = 209 nM (pChEMBL 6.679999828338623)
Descriptors: MW 393.42, ALogP 3.79, HBA 7, HBD 0, PSA 74.95, QED 0.5
Compound (SMILES): C=CC(=O)NCC(=O)N(C)c1cccc(NC(=O)c2cnc(NCCc3ccncc3)nc2NCCC)c1
InChIKey: NAELFTPQBUDKEC-UHFFFAOYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Enzyme Inhibition Assay: Quizartinib (Ambit Biosciences Corporation) was used as Reference Compound 1.6 μL (0.75 ng) of a mutant FLT3 protein (FLT3 D835Y, Life Technologies) and 3 μL of a solution (100 mmol/L HEPES, 10 mmol/L MgCl2, 25 mmol/L NaCl, 0.01% BSA, 1 mmol/L DTT, pH 7.5) containing a predetermined concentration of a test compound were mixed and allowed to incubate at 25° C. for 15 minutes. Thereafter, 3 μL (final concentration: 0.25 μmol/L) of a substrate peptide Biotin-AAA-AEEEEYFELVAKKK (Toray Industries, Inc.) (SEQ ID NO: 2) and 3 μL (final concentration: 15 μmol/L) of ATP (Sigma-Aldrich) were respectively added thereto, followed by shaking for 2 minutes and further allowing to incubate at 25° C. for 40 minutes to carry out an enzymatic reaction. It should be noted that FLT D835Y refers to an FLT3 protein with a substitution of aspartic acid 835 to tyrosine.Then, 30 μL of an enzyme reaction stop solution (5 μg/mL Streptavidin, 0.19 μg/mL PT66-K, 30 μmol/L HEPES (pH 7.0), 150 mmol/L KF, 75 mM EDTA, 0.15% BSA, 0.075% Tween20) containing Streptavidin-Xlent (Cisbio) and Mab PT66-K (Cisbio) was added to stop the enzymatic reaction, followed by allowing to incubate at room temperature for 1 hour to carry out an antigen-antibody reaction. Thereafter, phosphorylation of the substrate peptide was measured by measuring the time-resolved fluorescence of 615 nm and 665 nm using an Envision (PerkinElmer). By taking the value obtained by dividing the fluorescence value at 665 nm by the fluorescence value at 615 nm as a measured value, taking the measured value of the well with no addition of the compound (DMSO treatment only) and addition of ATP as 0% inhibition, and taking the measured value of the well with no addition of the compound (DMSO treatment only) and no addition of ATP as 100% inhibition, a 50% inhibitory concentration (IC50 value) of the test compound was calculated by Fit Model 205 of XLfit Ver. 5.3.1 (ID Business Solutions Limited).
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 516.61, ALogP 2.87, HBA 8, HBD 4, PSA 141.24, QED 0.25
Compound (SMILES): Cc1cc(F)c(C(=O)Nc2cccc(-c3nnnn3[C@H](C)CO)n2)cc1-c1cnn(C(C)C)c1
InChIKey: FCWQASHYRVZJOT-OAHLLOKOSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: HTRF KinEASE Assay: The ability (IC50) of compounds to inhibit ASK1 kinase activity was determined by HTRF® KinEASE Assay System. ASK1 was purchased from Thermofisher (Catalogue #PV4011), ATP was purchased from Sigma (Catalogue #A7699), HTRF® KinEASE Assay System was obtained from Cisbio (Bedford, Mass.). ½ Area plate was purchased from Perkin Elmer (Catalogue ##6005560). HTRF® KinEASE-STK is a generic method for measuring serine/threonine kinase activities using a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. The IC50 value for each compound was determined in the presence of compound (various concentration from 0 to 10 μM) and a fixed amount of ATP and peptide substrates. The test compound, 1 uM STK3 peptide substrate, and 5 nM of ASK1 kinase are incubated with kinase reaction buffer containing 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1 mM EGTA for 30 minutes. 100 uM ATP is added to start kinase reaction and incubated for 3 hours. The STK3-antibody labeled with Eu3+-Cryptate and 125 nM streptavidin-XL665 are mixed in a single addition with stop reagents provided by the Cisbio kit used to stop the kinase reaction. Fluorescence is detected using an Envision Multilabeled 2014 reader from PerkinElmer. The Fluorescence is measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm is calculated for each well.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 464.51, ALogP 3.43, HBA 9, HBD 2, PSA 123.64, QED 0.43
Compound (SMILES): CNCc1ccc(-n2cc(-c3nc(NC4CCN(S(C)(=O)=O)CC4)ncc3C(F)(F)F)cn2)c(C)c1
InChIKey: CPNJWSSSCAOSPW-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: CDK2/Cyclin E1 HERE Enzyme Activity Assay: CDK2/Cyclin E1 enzyme activity assays utilize full-length human CDK2 co-expressed as N-terminal GST-tagged protein with FLAG-Cyclin E1 in a baculovirus expression system (Cama Product Number 04-165). Assays are conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) is incubated with compounds (40 nL serially diluted in DMSO) in the presence of ATP (50 μM or 1 mM) and 50 nM ULight-labeled eIF4E-binding protein 1 (THR37/46) peptide (PerkinElmer) in assay buffer (containing 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.05 mg/ml BSA, and 0.01% Tween 20) for 60 minutes at room temperature. The reactions are stopped by the addition of EDTA and Europium-labeled anti-phospho-4E-BP1 antibody (PerkinElmer), for a final concentration of 15 mM and 1.5 nM, respectively. HTRF signals are read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data is analyzed with IDBS XLFit and GraphPad Prism 5.0 software using a three or four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 523.59, ALogP 3.21, HBA 8, HBD 2, PSA 105.04, QED 0.49
Compound (SMILES): N#C/C(=C\c1nc2c(N)ncnc2n1CCCO)C(N)=O
InChIKey: YADAJQRYASQJMB-QPJJXVBHSA-N
Target: Serine/threonine-protein kinase Nek2 (SINGLE PROTEIN), Homo sapiens
UniProt: P51955
Assay: Nek2 Inhibition Assay: For RSK2 kinase assays, see Example 64 above. Full-length human T175A NEK2 (referred to as NEK2) was expressed and purified as previously described (Knapp S. et al. J. Biol. Chem., 2007, 282: 6833-6842). The C22V mutant of NEK2 was generated by Quikchange mutagenesis (Stratagene) and was indistinguishable from NEK2 in kinase activity assays. NEK2 kinases (60 nM) in 20 mM Hepes, pH 7.6, 10 mM MgCl2, 1 mM EDTA, 0.2 mg/mL BSA, and 100 μM ATP were pre-incubated with inhibitors (8-10 concentrations, in duplicate) for 30 min at room temperature. Kinase reactions were initiated by the addition of 0.4 μCi/μL of γ-32P-ATP (6000 Ci/mmol, NEN) and 2.37 mg/mL β-casein (Sigma) and incubated for 30 minutes at room temperature.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 287.28, ALogP -0.82, HBA 8, HBD 3, PSA 156.73, QED 0.48
Compound (SMILES): O=C(c1cscn1)N1C2CCC1CN(Cc1c(-c3ccc(Cl)cc3)nc3ncccn13)C2
InChIKey: PUGCCFFAORWRLJ-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: In Vitro Electrophysiological Assay: Table 1: In Vitro Electrophysiological Analysis of the Human TASK-1 and TASK-3 Channels Via Two-Electrode Voltage Clamp Technique in Xenopus laevis Oocytes. Xenopus laevis oocytes were selected as described elsewhere by way of illustration [Decher et al., FEBS Lett. 492, 84-89 (2001)]. Subsequently, the oocytes were injected with 0.5-5 ng of a cRNA solution coding for TASK-1 or TASK-3. For the electrophysiological analysis of the channel proteins expressed in the oocytes, the two-electrode voltage clamp technique [Stühmer, Methods Enzymol. 207, 319-339 (1992)] was used. The measurements were conducted as described [Decher et al., FEBS Lett. 492, 84-89 (2001)] at room temperature (21-22° C.) using a Turbo TEC 10CD amplifier (NPI), recorded at 2 kHz and filtered with 0.4 kHz. Substance administration was performed using a gravitation-driven perfusion system. Here, the oocyte is located in a measuring chamber and exposed to the solution stream of 10 ml/min. The level in the measuring chamber is monitored and regulated by sucking off the solution using a peristaltic pump.
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 464.98, ALogP 4, HBA 7, HBD 0, PSA 66.63, QED 0.46
Compound (SMILES): CC(C)(C#Cc1cc(C(F)(F)F)ccc1CN1CCN(C(=O)OC(C(F)(F)F)C(F)(F)F)CC1)OC=O
InChIKey: OOYGNEIRGHQJTD-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Competitive Activity-Based Protein Profiling (Human): Proteomes (human prefrontal cortex or cell membrane fractions) (50 μL, 1.0-2.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FP-Rh or JW912 (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at room temperature. Reactions were quenched with SDS loading buffer (15 μL 4x) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL using ImageJ 1.49k software.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 548.4, ALogP 4.76, HBA 5, HBD 0, PSA 59.08, QED 0.3
Compound (SMILES): COc1cc(OC)nc(O[C@@H]2CC[C@@H](C)N(C(=O)c3ccccc3-n3nccn3)C2)c1
InChIKey: MQRFEOYYAKWIDK-HZPDHXFCSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well.
Measurement: IC50 = 71 nM (pChEMBL 7.150000095367432)
Descriptors: MW 423.47, ALogP 2.75, HBA 8, HBD 0, PSA 91.6, QED 0.6