lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nsc(NC(=O)C2CCCCC2C(=O)c2ccc(-c3ccn[nH]3)cc2)c1C#N
InChIKey: PXTLAIJIMSKGJS-UHFFFAOYSA-N
Target: Polyunsaturated fatty acid 5-lipoxygenase (SINGLE PROTEIN), Homo sapiens
UniProt: P09917
Assay: FLAP Binding Assay (Test A): Compounds were tested in a competition binding assay using 3H-MK591 as tracer. (Preparation of MK-591 is described in Bioorg. Med. Chem. Lett. 1999, 9, 2391). A 100,000×g pellet from COS-7 cells stably transfected with a plasmid expressing human ALOX5AP was the source of FLAP. Membrane pellets were resuspended in buffer (100 mM Tris-HCl, 0.05% Tween-20, 140 mM NaCl, 2 mM EDTA, 0.5 mM DTT, 5% Glycerol, pH 7.5) to give a final protein concentration of 12 mg/mL (2 μg/well). To perform assays, 1.4 μL compounds were dispensed into 96-well plates in 3-fold dilution series in triplicate. 84 μL radioligand (25000 CPM, 2 nM final concentration in assay) was then added followed by 84 μL membrane suspension and incubation at rt for 60 min. Following filtration, filter plates were dried 12 h at RT (or 50° C. for 1 hour). 50 μL scintillant was then added, the filterplates were sealed and radioactivity was measured in a microbeta counter. Specific binding was defined as total binding minus non-specific binding. Total binding was defined as 3H-MK591 bound to membranes in the absence of competitor, non-specific binding was defined as 3H-MK591 in the presence of 0.1 mM MK-591. IC50 values were determined by plotting % inhibition versus log compound concentration and using a one site dose response model.
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 419.51, ALogP 4.34, HBA 6, HBD 2, PSA 111.53, QED 0.6
Compound (SMILES): COc1cc2ncnc(Nc3ccc(NC(=O)Nc4ccc(Oc5ccccc5)cc4)c(Cl)c3)c2c2c1OCCO2
InChIKey: JQCJVVXZUDGXCR-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Test of Small Molecule Compounds for Inhibiting the Activity of VEGFR-2 Kinase: 1. Dilution of the compound: a total of 12 concentrations were obtained using a 4-fold gradient dilution from the highest concentration of 10000 nM (the maximum final concentration of the drug used in this experiment is 10000 nM, and the minimum final concentration is 0.002384 nM),2. 2.5 μl of the gradient-diluted compounds was taken with a transfer pipette to a 384-well plate,3. Addition of enzyme: 5 μl of 2×VEGFR-2 kinase was taken with a transfer pipette to the corresponding reaction well of the 384-well plate, which was mixed and pre-reacted at room temperature for 30 min,4. 2.5 μl of 4× substrate/ATP Mix was taken with a transfer pipette to the corresponding reaction well of the 384-well plate,5. Negative control: 2.5 μl/well 4× substrate/ATP Mix and 7.5 μl 1× Kinase Assay Buffer were added to the wells of the 384-well plate,Positive control: 2.5 μl/well 4× substrate/ATP Mix, 2.5 μl/well 1× Kinase Assay Buffer containing 4% DMSO, and 5 μl/well 2×VEGFR-2 solution were added to the 384-well plate. The final concentration of DMSO in the reaction system is 4%,6. The mixture was mixed well and then centrifuged and reacted at room temperature in dark for 60 min,7. Termination of the enzymatic reaction: 5 μl of 4× Stop solution was taken with a transfer pipette to the wells of the 384-well plate, mixed and then centrifuged, and reacted at room temperature for 5 min,8. Development of the reaction: 5 μl of 4× Detection Mix was taken with a transfer pipette to the wells of the 384-well plate for color development, and the mixture was mixed and then centrifuged and reacted at room temperature for 60 min,9. The 384-well plate was placed into the Envision plate reader and the signal was detected using the appropriate program,10. Analysis and processing of the raw data:The drug concentrations and the corresponding inhibition rates were input into GraphPad Prism5 for calculation, and the inhibition rate of the compound was calculated as follows: inhibition rate (%)=[1−(experimental well reading value−negative control well reading value)/(positive control well reading value−negative control well reading value)]×100%. Processing with GraphPad Prism5 software yielded the corresponding IC50 value (the concentration of the compound at which 50% of the highest inhibition of the enzyme is achieved).
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 570.01, ALogP 7.24, HBA 8, HBD 3, PSA 115.86, QED 0.19
Compound (SMILES): O=C(Nc1ccc(Br)cc1F)N[C@@H](Cc1ccccc1)C(=O)NCCO
InChIKey: ISXHVEOMRHCILC-INIZCTEOSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FLIPR: HEK-Gα16 cells stably expressing the human FPR2 receptor was utilized. Cells were plated into 384-well poly-D-lysine coated plates at a density of 18,000 cells per well one day prior to use. The growth media was DMEM medium supplemented with 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic, 50 μg/ml hygromycin, and 400 μg/ml geneticin. On the day of the experiment, the cells were washed twice with Hank's Balanced Salt Solution supplemented with 20 mM HEPES (HBSS/hepes buffer). The cells were then dye loaded with 2 μM Fluo-4 diluted in the HBSS/Hepes buffer and incubated at 37° C. for 40 minutes. Extracellular dye was removed by washing the cell plates four times prior to placing the plates in the FLIPR (Fluorometric Imaging Plate Reader, Molecular Devices). Ligands were diluted in HBSS/Hepes buffer and prepared in 384-well microplates.
Measurement: EC50 = 15600 nM (pChEMBL 4.809999942779541)
Descriptors: MW 424.27, ALogP 2.43, HBA 3, HBD 4, PSA 90.46, QED 0.55
Compound (SMILES): COC(=O)c1cccc(C(=O)C2CCN(C(=O)C[C@@H](C)Cc3nc4c(C)cccc4c(=O)[nH]3)CC2)c1
InChIKey: MOMSZDNLTDTPMD-KRWDZBQOSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Biochemical Activity : PARP-1: The autoparsylation assay is performed as 384-well HTRF® (Cisbio, Codolet, France) assay format in Greiner low volume nb 384-well microtiter plates. 35 nM His-tagged Parp-1 (human, recombinant, Enzo Life Sciences GmbH, Lörrach, Germany) and a mixture of 125 nM bio-NAD (Biolog, Life science Inst., Bremen, Germany) and 800 nM NAD as co-substrate are incubated in a total volume of 6 μl (100 mM Tris/HCl, 4 mM Mg-chloride, 0.01% IGEPAL® CA630, 1 mM DTT, 0.5% DMSO, pH 8, 13 ng/μl activated DNA (BPS Bioscience, San Diego, US)) in the absence or presence of the test compound (10 dilution concentrations) for 150 min at 23° C. The reaction is stopped by the addition of 4 μl of the Stop/detection solution (70 nM SA-Xlent® (Cisbio, Codolet, France), 2.5 nM Anti-His-K® (Eu-labelled anti-His, Cisbio, Codolet, France) in 50 mM HEPES, 400 mM KF, 0.1% BSA, 20 mM EDTA, pH 7.0). After 1 h incubation at room temperature the HTRF iss measured with an Envision multimode reader (Perkin Elmer LAS Germany GmbH) at excitation wavelength 340 nm (laser mode) and emission wavelengths 615 nm and 665 nm. The ratio of the emission signals is determined. The full value used is the inhibitor-free reaction. The pharmacological zero value used is Olaparib (LClabs, Woburn, US) in a final concentration of 1 μM.
Measurement: IC50 = 790 nM (pChEMBL 6.099999904632568)
Descriptors: MW 489.57, ALogP 3.71, HBA 6, HBD 1, PSA 109.43, QED 0.4
Compound (SMILES): CO[C@H]1CC[C@H](C(=O)N2Cc3cccnc3Nc3ccc(N4CCOCC4C4CC4)cc32)CC1
InChIKey: XBLKAWRXJMMVDM-DOABJOQISA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Enzymatic Assay: Each test compound (10 mM stock in DMSO) is diluted in DMSO to make a 10-point, 3-fold dilution series. 125 nL of each dilution or DMSO alone is dispensed to a 384-well Greiner Lumitrac 200 assay plate using an Echo® Liquid Handler. To each well of the plate is added 20 uL of enzyme in assay buffer or assay buffer alone. Assay buffer consists of 50 mM sodium phosphate, pH 7.0, 50 mM magnesium chloride, 50 mM sodium chloride, and 0.01% (w/v) bovine serum albumin. When present, the R132H mutant IDH1 enzyme is at a working concentration of 1.875 nM (final concentration in assay of 1.5 nM). The assay plate is allowed to incubate for 30 minutes at room temperature and 5 uL of 5× substrate mixture (2.5 uM nicotinamide adenine dinucleotide phosphate, 100 uM adenosine diphosphate, 7.5 mM glyceraldehyde-3-phosphate, 7.5 ug/mL of spinach glyceraldehyde-3-phosphate dehydrogenase, 25 nM phosphoglycerate kinase, and 5 mM alpha-ketoglutarate in assay buffer) is added to all wells. The reaction plate is incubated for 60 minutes followed by addition of 25 uL of Promega Kinase-GLO reagent to all wells and 10-minute incubation.Luminescence is measured using a PerkinElmer Envision plate reader. The percent activity of each dilution is determined as the ratio of background corrected signal to the background corrected signal of wells receiving only DMSO. IC50 values are determined by fitting percent activity data to a four-parameter logistic dose response equation.
Measurement: IC50 = 428 nM (pChEMBL 6.369999885559082)
Descriptors: MW 462.59, ALogP 4.49, HBA 6, HBD 1, PSA 66.93, QED 0.72
Compound (SMILES): Cn1cc(-c2cccc(C(=O)Nc3ccc4nn(CC(=O)N5CCC(C(C)(C)O)CC5)cc4c3)n2)cn1
InChIKey: WCRXXVUUUJTTPR-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: For the assay, 11 different concentrations in the range from 20 μM to 0.073 nM were prepared from a 2 mM solution of the test substance in DMSO. For the assay, 50 nl of the respective solution were pipetted into a black low-volume 384-well microtitre plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of Irak4 in assay buffer [50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 30 μM activated sodium orthovanadate, 0.1% (w/v) of bovine gamma-globulin (BGG) 0.04% (v/v) nonidet-P40 (Sigma)] were added and the mixture was incubated for 15 min to allow prebinding of the substances to the enzyme prior to the kinase reaction. The kinase reaction was then started by addition of 3 μl of a solution of adenosine triphosphate (ATP, 1.67 mM=final concentration in 5 μl of assay volume: 1 μM) and peptide substrate (0.83 μM=final concentration in 5 μl assay volume: 0.5 μM) in assay buffer, and the resulting mixture was incubated at 22° C. for the reaction time of 45 min. The concentration of the Irak4 was adjusted to the respective activity of the enzyme and set such that the assay was carried out in the linear range. Typical concentrations were in the order of about 0.2 nM. The reaction was stopped by addition of 5 μl of a solution of TR-FRET detection reagents [0.1 μM streptavidin-XL665 (Cisbio Bioassays; France, catalogue No. 610SAXLG) and 1.5 nM anti-phosphoserin antibody [Merck Millipore, STK Antibody, catalogue No. 35-002] and 0.6 nM LANCE EU-W1024-labelled anti-mouse-IgG antibody (Perkin-Elmer, product No. AD0077, alternatively it is possible to use a terbium cryptate-labelled anti-mouse-IgG antibody from Cisbio Bioassays) in aqueous EDTA solution (100 mM EDTA, 0.4% [w/v] bovine serum albumin [BSA] in 25 mM HEPES pH 7.5).
Measurement: IC50 = 142 nM (pChEMBL 6.849999904632568)
Descriptors: MW 501.59, ALogP 3.09, HBA 8, HBD 2, PSA 118.17, QED 0.42
Compound (SMILES): OC(c1cncc(-c2ccc3nc(-c4nc5ccccc5[nH]4)oc3c2)c1)C(F)(F)F
InChIKey: IBIJVGHFFIMZAD-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Scintillation Proximity Assay (PI3K-γ): [γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kγ (p110γ) Recombinant Human Protein was purchased from Life technology (Grand Island, N.Y.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer).
Measurement: IC50 < 250 nM (pChEMBL 6.6020002365112305)
Descriptors: MW 410.36, ALogP 5.03, HBA 5, HBD 2, PSA 87.83, QED 0.44
Compound (SMILES): CCOc1nn(-c2ccccc2)c(NC(=O)N[C@@H]2CN(CCOC)O[C@H]2c2ccc(F)c(F)c2)c1C
InChIKey: MDPARYBRPSJSPK-YADHBBJMSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Assay: Seven point five μL per well of human TrkA (PV3144, Lifetechnologies, final concentration: 1 nmol/L) suspended in the assay buffer (100 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20 and 1 mmol/L dithiothreitol (DTT)) was applied in a 384 well plate and the plate was pre-incubated for 15 min at room temperature with 0.4 μL of each compound (final concentration: 200 μmol/L-1 pmol/L) dissolved in DMSO. Then, fluorescent substrate (FL-peptide 27, 760424, PerkinElmer, final concentration: 1.5 μmol/L) and ATP (final concentration 500 μmol/L) dissolved in the assay buffer was added in each well. After the incubation of 120 min at 37° C., fifteen μL of termination buffer (100 mmol/L HEPES, 40 mmol/L ethylenediaminetetraacetic acid (EDTA), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20, 1 mmol/L DTT and 0.16 vol % Coating Reagent 3) was added in each well to stop the enzyme reaction. Fluorescent intensities (FI) of phosphorylated and non-phosphorylated fluorescent substrates were measured by LabChip EZReader II (Caliper LifeSciences, Inc.), and conversion ratio (CR) was calculated by the following formula-1. The CR in the well applied with DMSO alone was used as a negative control and the CR in the well without applying TrkA was used as a positive control.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 501.53, ALogP 3.98, HBA 7, HBD 2, PSA 89.88, QED 0.46
Compound (SMILES): COc1cnc2c(-c3nc4cc(F)c(O[C@H]5CCC[C@H]5OC(=O)Nc5cnc(C)nc5)cc4s3)cc(C)cc2n1
InChIKey: DTZZPVZZVKJMFH-FCHUYYIVSA-N
Target: Proteinase-activated receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI0
Assay: FLIPR Assay in PAR4-Expressing HEK293 Cells: FLIPR-based calcium mobilization assay in HEK293 cells was used to measure PAR4 antagonism agonism, and selectivity against PAR1. The activity of the PAR4 antagonists of the present invention were tested in PAR4 expressing cells by monitoring H-Ala-Phe(4-F)-Pro-Gly-Trp-Leu-Val-Lys-Asn-Gly-NH2-induced intracellular calcium mobilization. Counter screens for agonist activity and PAR1 antagonist activity were also performed. Briefly, PAR/PAR4-expressing HEK293 cells were grown in DMEM (Life Technology, Grand Island, N.Y.) containing 10% heat-inactivated FBS, 1% Penicillin-Streptomycin, 10 μg/mL blasticidin, and 100 μg/mL Zeocin at 37° C. with 5% CO2. Cells were plated overnight prior to the experiment in a black 384-well Purecoat Amine clear bottom plate (Becton Dickinson Biosciences, San Jose, Calif.) at 10,000 cells/well in 30 μL growth medium and incubated in a humidified chamber at 37° C. with 5% CO2 overnight. Prior to compound addition, the cell medium was replaced with 40 μL of 1× calcium and magnesium-containing Hank's Balanced Saline Solution (HBSS) (with 20 mM HEPES) and 1:1000 diluted fluorescent calcium indicator (Codex Biosolutions, Gaithersburg, Md.). After a 30 minute incubation period at 37° C. and a further 30 minute incubation and equilibration period at room temperature, 20 μL test compound (diluted in 1×HBSS buffer) was added at various concentrations at 0.17% dimethyl sulfoxide (DMSO) final concentration. Changes in fluorescence intensity were measured using a Functional Drug Screening System (FDSS, Hamamatsu, Japan) to determine agonist activities. The cells were then incubated for 30 minutes at room temperature followed by addition of 20 μL of agonist peptide for antagonist activity measurement. The PAR4 agonist peptide (H-Ala-Phe(4-F)-Pro-Gly-Trp-Leu-V al-Lys-Asn-Gly-NH2) and the PAR1 agonist peptide (SFFLRR) were routinely tested to ensure a proper response at the EC50 value in the assay (5 μM for PAR4 agonist peptide and 2 μM for PAR1 agonist peptide). Compound potency was derived from 11-point concentration-response curves.
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 560.61, ALogP 6.01, HBA 10, HBD 1, PSA 121.24, QED 0.27
Compound (SMILES): Cc1nc2ccc(/N=c3\[nH]c(=O)n(CCC(=O)O)c(=O)n3CC3CCC(C)CC3)cc2s1
InChIKey: SJZITDUYBKNGQA-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% 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, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% 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, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 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, 50 nM ATP solution (50 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 3 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. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 457.56, ALogP 2.79, HBA 8, HBD 2, PSA 122.34, QED 0.59
Compound (SMILES): Cc1ncc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1N1C(=O)C(C)(C)OC[C@@H]1C
InChIKey: LDFAOKLVLKOKNG-JTQLQIEISA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assays: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 434.42, ALogP 3.23, HBA 7, HBD 1, PSA 98.64, QED 0.67
Compound (SMILES): COc1ccc(C(=O)N2CC3(CCN(C(=O)C#Cc4ccc(F)cc4)CC3)C2)cc1
InChIKey: VTBOGXRTCDBPAB-UHFFFAOYSA-N
Target: G-protein coupled receptor 183 (SINGLE PROTEIN), Homo sapiens
UniProt: P32249
Assay: Cell-Based Luminescent Assay: The test compounds that demonstrated a corrected % activity of >=50% were defined as inhibitors of the reaction. The experimental values were normalized by the difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc.
Measurement: IC50 = 12.5 nM (pChEMBL 7.900000095367432)
Descriptors: MW 406.46, ALogP 2.95, HBA 3, HBD 0, PSA 49.85, QED 0.72
Compound (SMILES): CC(=O)c1nn(CC(=O)N2C(C(=O)Nc3nc(Br)ccc3C)C[C@@]3(C)C[C@@H]23)c2c(C3CC3)cc(-c3cnc(C)nc3)cc12
InChIKey: HSMRPYPHPQDHLG-QWEUKOSNSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: 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-SBz 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 < 1000 nM (pChEMBL 6)
Descriptors: MW 642.56, ALogP 5.37, HBA 8, HBD 1, PSA 122.97, QED 0.21
Compound (SMILES): C=CC(=O)N1CC(NC(=O)Cn2cc(C(=O)NCCC(=O)Nc3ccccc3CC)c3cc(Br)cc(C)c32)C1
InChIKey: IRKRMMLCLMQFRQ-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Coupled Nucleotide Exchange Assay (2 hours): Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 2 hours. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 430 nM (pChEMBL 6.369999885559082)
Descriptors: MW 594.51, ALogP 3.55, HBA 5, HBD 3, PSA 112.54, QED 0.31
Compound (SMILES): N#Cc1c(N)nn2ccc(N3C[C@@H](O)C[C@@H]3c3cc(F)ccc3F)nc12
InChIKey: OYWAUYQPEXLVTP-IINYFYTJSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibitory Activity Assay: TrkA, TrkB and TrkC:A testing platform for TrkA, TrkB and TrkC kinase activity was established based on Homogeneous Time-Resolved Fluorescence (HTRF) assay, and the activities of the compounds were tested using the platform. The compounds were subjected to three-fold gradient dilution with 100% DMSO with a starting concentration of 1 mM (11 concentrations in total). 4 μL of diluted sample for each concentration was added to 96 μL of reaction buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM NaVO3, 0.001% Tween-20, 0.01% BSA and 1 mM DTT) and mixed homogeneously to be used as a 4* compound. The reaction buffer was used to formulate 2* TrkA, TrkB and TrkC kinases (purchased from Carna Biosciences 08-186, 08-187, 08-197, and the final concentrations thereof were 0.5 nM, 0.1 nM, and 1 nM, respectively) and 4* substrate mixture (ATP+TK peptide) (wherein the final concentrations of ATP were 40 μM, 50 μM, and 20 μM, respectively; TK peptide, HTRF® KinEASE-TK, was purchased from Cisbio, and the final concentration thereof was 100 nM) for use. 2.5 μL of the 4* compound was added to a 384-well plate (OptiPlate-384, purchased from PerkinElmer), and then 5 μL of the 2* TrkA, TrkB and TrkC kinases were added, and mixed homogeneously by centrifugation. Then 2.5 μL of the 4* substrate mixture was added to initiate the reaction (the total reaction volume is 10 μL). The 384-well plate was placed in an incubator and incubated for 60 min at 23° C. Then the reaction was terminated by adding 5 μL of Eu3+ cryptate-labeled anti-phosphotyrosine antibody (HTRF® KinEASE-TK, purchased from Cisbio), and 5 μL of Streptavidin-XL-665 (HTRF® KinEASE-TK, purchased from Cisbio). After incubated for 1 hr in the incubator, the fluorescence values were read on Envision (purchased from PerkinElmer).
Measurement: IC50 < 25 nM (pChEMBL 7.6020002365112305)
Descriptors: MW 356.34, ALogP 1.77, HBA 7, HBD 2, PSA 103.47, QED 0.73
Compound (SMILES): COc1nn(C)c(C)c1S(=O)(=O)N1C[C@H]([C@H](C)NC(C)=O)Oc2ccc(NC(=O)OC(C)(C)C(F)(F)F)cc21
InChIKey: NDUGGHDMMADLQQ-KPZWWZAWSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biochemical TR-FRET assay: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., a biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 μg/mL bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per μl in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 μl SF9 lysate per 15 μl of diluted RORγ-LBD.
Measurement: IC50 = 61.3 nM (pChEMBL 7.210000038146973)
Descriptors: MW 577.58, ALogP 3.11, HBA 9, HBD 2, PSA 141.09, QED 0.51
Compound (SMILES): CSc1ccc(C(=O)N2CCc3ccc(Nc4nc(N[C@@H]5CCCC[C@@H]5N)ncc4C(N)=O)cc3C2)cc1
InChIKey: ZNSZZPFGAWFUPU-BJKOFHAPSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: ADP-Glo Kinase Assay: The Promega ADP-Glo Kinase Assay is a luminescent ADP detection assay that provides a universal, homogeneous, high-throughput screening method to measure kinase activity by quantifying the amount of ADP produced during a kinase reaction.
Measurement: IC50 = 37.4 nM (pChEMBL 7.429999828338623)
Descriptors: MW 531.69, ALogP 3.92, HBA 8, HBD 4, PSA 139.26, QED 0.34
Compound (SMILES): CN(C)C(=O)/C=C/CC[C@H](OC(=O)N(C)C)C(=O)Nc1cccn(Cc2nc3cc(F)ccc3n2CC(C)(C)C)c1=O
InChIKey: PJLGZLODKGDWMY-JEAXJQIYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 μM), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 μM final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 μl/well termination buffer (Carna Biosciences) and signal determination using a Caliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 = 2750 nM (pChEMBL 5.559999942779541)
Descriptors: MW 582.68, ALogP 3.86, HBA 8, HBD 1, PSA 118.77, QED 0.36
Compound (SMILES): Cc1ccc2[nH]ncc2c1-c1ccc2nc(NC(=O)[C@@H]3C[C@@H]3F)cn2c1
InChIKey: QSJNDHJHZPSKFF-OCCSQVGLSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: ADP-Glo kinase assay: HPK1 kinase activity was measured by Promega's ADP-Glo kinase assay. In this assay, 5 ng of recombinant human HPK1 (signalchem) is incubated with 5 μL of compounds (0.5% DMSO), 5 μL of MBP (0.5 μg/μl) and 5 μL of ATP (25 μM) in buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg/ml BSA; 5 μM DTT). The assay was started by incubating the reaction mixture in a 96-well plate at 30′ C for 40 minutes. After the incubation, 25 uL ADP-Glo reagent was added and the reaction was incubated at room temperature for 40-min to stop the reaction and degrade residual ATP. The ADP product was then converted to ATP by adding 50 uL per well of detection reagent. Luminescence was detected after 30-min room temperature incubation with the Molecular device I3X plate reader. The IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentration using software routines as implemented in the GraphPad Prism 7 software and SigmaPlot13.0.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 349.37, ALogP 3.48, HBA 4, HBD 2, PSA 75.08, QED 0.59
Compound (SMILES): CC(=O)N[C@@H](C)CO[C@H]1C[C@H](COc2ncnc3c2ccn3Cc2cccc(F)c2)C1
InChIKey: DSVUYULCESKBJC-QSFXBCCZSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Inhibitory Activity on Human ACC1 and the ACC2: Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H]−+Intensity of [AcCoA-H]−) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
Measurement: IC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 426.49, ALogP 3.32, HBA 6, HBD 1, PSA 78.27, QED 0.57
Compound (SMILES): CC(C)S(=O)(=O)c1ccc(-c2cnc(N)c(-c3nnc(-c4ccc(CN[C@H](C)CO)cc4F)o3)n2)cc1
InChIKey: VTZLKDZUOKGUPG-OAHLLOKOSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 M [γ-33P]ATP (3mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).The reaction was stopped after 24 hours by the addition of 30 μL 0.1M phosphoric acid containing 2 mM ATP. A multiscreen phosphocellulose filter 96-well plate (Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2M phosphoric acid prior to the addition of 45 μL of the stopped assay mixture. The plate was washed with 5×200 μL 0.2M phosphoric acid. After drying, 100 μL Optiphase SuperMix liquid scintillation cocktail (Perkin Elmer) was added to the well prior to scintillation counting (1450 Microbeta Liquid Scintillation Counter, Wallac).
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 526.59, ALogP 3.23, HBA 10, HBD 3, PSA 157.12, QED 0.3
Compound (SMILES): COc1cccc(CN2CC[C@H](N(C)c3cc(F)c(S(=O)(=O)Nc4cscn4)cc3Cl)C2)n1
InChIKey: SYIMLQDKCUQRPH-HNNXBMFYSA-N
Target: Sodium channel protein type 8 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQD0
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS ρCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 3197 nM (pChEMBL 5.5)
Descriptors: MW 512.03, ALogP 3.85, HBA 8, HBD 1, PSA 87.66, QED 0.49
Compound (SMILES): O=c1[nH]ncc(N2Cc3cccc(NC4CCCOC4)c3C2)c1C(F)(F)F
InChIKey: PJWTVMOOSHGQBG-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 JAL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision plate reader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 380.37, ALogP 2.9, HBA 5, HBD 2, PSA 70.25, QED 0.86
Compound (SMILES): COc1cc(CO)c(-c2cn3ccc(N4CCC(NS(=O)(=O)C5CC5)CC4)cc3n2)cc1Cl
InChIKey: ANQNTDVTIJGMMI-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase SUV39H2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H5I1
Assay: Suv 39H2 Assay: Assay buffer (4 μL) was added into all the wells, including test compound and control wells (except for Sinefungin control wells), using the Multidrop Combi. The plate was centrifuged at 1000 rpm for 1 min. Substrate mix (8 μL) containing radiolabelled 3H-SAM (final conc: 100 nM) and H3 Histone Peptide (final conc: 350 nM) was added to all wells using Multidrop Combi and centrifuged at 1000 rpm for 1 min. SUV39H2 (8 μL, final conc: optimized for each lot of the enzyme based on specific activity) was added by using the Multidrop Combi. The assay plate was centrifuged at 1000 rpm for 1 min. Assay buffer was used as background control, and incubated at room temperature for 3 hours. The reaction was stopped with 20 μL of 2.5 mg/mL Streptavidin SPA beads (final conc. 50 μg/well) in buffer using the Multidrop Combi and centrifuged for 1 min. The plate was loaded into the Trilux-Microbeta counter and a delayed read of 10 hours was made. The radioactive signal was measured at 1 min/well. Sinefungin and the internal standard compound were used as tool compounds to determine assay performance.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 491.01, ALogP 3.21, HBA 7, HBD 2, PSA 96.17, QED 0.53
Compound (SMILES): CN(CCCO)c1ccc2ncc(-c3cc4ccccc4o3)n2n1
InChIKey: WKFWYZWHYZPDOE-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: Kinase Assay: A recombinant fusion protein of Glutathione-S-Transferase (GST, N-terminally) and human full-length MKNK1 (amino acids 1-424 and T344D of accession number BAA 19885.1), expressed in insect cells using baculovirus expression system and purified via glutathione sepharose affinity chromatography, was purchased from Carna Biosciences (product no 02-145) and used as enzyme. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-IKKRKLTRRKSLKG (C-terminus in amide form) was used which can be purchased e.g. form the company Biosyntan (Berlin-Buch, Germany).For the assay 50 nL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM magnesium chloride, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μL assay volume is 10 μM) and substrate (0.1 μM=>final conc. in the 5 μL assay volume is 0.06 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 45 min at 22° C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.05 μg/ml. The reaction was stopped by the addition of 5 μL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein 56 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 322.37, ALogP 2.96, HBA 6, HBD 1, PSA 66.8, QED 0.61
Compound (SMILES): Nc1ccc(-c2cccs2)cc1NC(=O)N1CCC1
InChIKey: DAOGDASBGHDEMN-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Inhibition of Histone Deacetylase Enzymatic Activity Assay: HDAC1: The following trypsin-coupled protocol and Caliper protocol described Herein was used to assay the compounds of the invention. Trypsin Coupled Assay: HDAC1-Kinetic IC50 3Hr PreIncubation. Working Stocks: 1xHDAC Assay Buffer: 50 mM HEPES (GIBCO 15630-114), 100mM KCl, 0.001% Tweem-20 (Zymed 00-3005), 0.05% BSA (Invitrogen, P2489), pH 7.4, 1.5xHDAC+1.5xTrypsin: 225nM Trypsin(Worthinton)+0.18 ug/ml HDAC1 (BPS Inc) in assay buffer 300x Trypsin stock is made up in HDAC Assay Buffer, aliquoted and stored at -80C. 3xSubstrate: 18uM Substrate in assay buffer BPS Enzyme and Substrate amounts (per well).
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 273.36, ALogP 3.23, HBA 3, HBD 2, PSA 58.36, QED 0.83
Compound (SMILES): NC/C(=C\F)COc1ccc(S(=O)(=O)CC23CC(C(=O)NC4CCC4)(C2)C3)cc1
InChIKey: LMECYWAKXCUVEY-OVCLIPMQSA-N
Target: Amine oxidase [copper-containing] 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75106
Assay: SSAO Activity Assay: Amine oxidase activity of recombinant hSSAO, hMAO-A, and hMAO-B isoforms are measured using the MAO-Glo assay kit (Promega, V1402). Ten nL test compounds (with DMSO as vehicle, 0.1% v/v for hSSAO, hMAO-A and hMAO-B) were added into assay plates by Echo, and pre-incubated with 5 μL of enzyme for 30 mins at room temperature before the addition of 5 μL of luminogenic substrate. The substrate concentration is 40 μM for human recombinant SSAO, 40 μM for human recombinant MAO-A, and 4 μM for human recombinant MAO-B. The hSSAO and hMAO-A assays were conducted in the MAO-A reaction buffer in the kit, and the MAO-B assay was conducted in the MAO-B reaction buffer. Oxidation of the substrate was conducted for 1 hr, before the addition of detecting reagent according the manufacture's protocol. The IC50 value of the tested compounds was calculated by fitting the dose response curve using a 4-parameter non-linear regression routine.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 422.52, ALogP 2.49, HBA 5, HBD 2, PSA 98.49, QED 0.64
Compound (SMILES): CC(=O)c1nn(CC(=O)N2[C@H]3C[C@@]4(CCCCCCCOCc5ccc(Br)nc5NC3=O)C[C@@H]24)c2cnc(-c3cnc(C)nc3)cc12
InChIKey: AQTPNQBDJZTTNA-HOFKBBIZSA-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 701.63, ALogP 5.43, HBA 10, HBD 1, PSA 145.09, QED 0.22
Compound (SMILES): COc1ccc(CN2CCn3nnnc3C2c2cccc(F)c2)c(OC)c1
InChIKey: PUESEFPVUAPOKF-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: TR-FRET Assay: In this assay, a GST-tagged human RORγ ligand binding domain (GST-hRORγ-LBD) interacts with a synthetic biotinylated TRAP220 cofactor peptide containing an LXXLL motif (amino acids 631-655 from NP_004765). The ligand-binding domain (LBD) of RORγ is expressed as fusion protein with GST in BL-21 (BL3) cells using the vector pDEST15. Cells are lysed by lysozyme-treatment and sonication, and the fusion proteins purified over glutathione sepharose (Pharmacia) according to the manufacturers instructions. The strong constitutive interaction is disrupted upon binding of functional antagonists. The strength of the interaction is monitored by TR-FRET between streptavidine APC interacting with the biotinylated peptide and Europium-labelled Anti-GST. 6 μl RORγ-LBD-GST solution (final 8.7 nM) in assay buffer (20 mM Tris-HCl, pH 6.8, 5 mM MgCl2, 60 mM KCl, 0.1% delipidated BSA, 1 mM DTT) are dispensed into black 384 well small volume plates (Greiner #784076). 6 μl TRAP220 peptide (final 400 nM), SA-APC (final 1.6 ng/μl), Eu-anti-GST (final 0.125 ng/μl) in assay buffer and 2 μl test compounds (DMSO stocks prediluted in 20 mM Tris-HCl, pH 6.8, 5 mM MgCl2, 60 mM KCl) are added and the mixture incubated 60 min at RT in the dark before measuring FRET (excitation 337 nm, emission at 615 and 665 nm).
Measurement: IC50 = 5445 nM (pChEMBL 5.260000228881836)
Descriptors: MW 369.4, ALogP 2.43, HBA 7, HBD 0, PSA 65.3, QED 0.69