lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccccc1-c1cc(=O)n(C)cc1-c1cnn(Cc2ccccc2)c1
InChIKey: ZNVUXNJXHZSJRK-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: In Vitro CBP Inhibition: The CBP-inhibitory activity of the compounds described herein was determined by calculating the IC50. More specifically, CBP inhibitor activity was assayed as follows: CBP was cloned and expressed in E. coli as His-tag protein and purified by Nickel affinity and gel-filtration chromatography. The protein was further characterized as a single band with the correct molecular weight by SDS-PAGE. CBP binding and inhibition is assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Perkin Elmer). In a 384-well ProxiPlate CBP (50 nM final) was combined with peptide (20 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 min incubation at room temp, Alpha-streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 2750 nM (pChEMBL 5.559999942779541)
Descriptors: MW 355.44, ALogP 4.27, HBA 4, HBD 0, PSA 39.82, QED 0.55
Compound (SMILES): C.C[C@H](CO)n1cnnc1-c1cccc(NC(=O)c2cc(CC(=O)c3ccccc3)ccc2F)n1
InChIKey: IVLIFGPMGGIMEY-PKLMIRHRSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: ADP-Glo Assay: Table 3: The ability of test compounds to inhibit ASK1 kinase activity was determined using the luminescent ADP-Glo Kinase Assay and Myelin Basic Protein (MBP) as a substrate (Promega Corporation, Madison, Wis.). During the kinase reaction, ASK1 utilizes ATP and generates ADP. The remaining ATP is then depleted by addition of the ADP-Glo reagent which also terminates the reaction. Subsequent addition of the Kinase Detection Reagent converts the ADP that was produced during the kinase reaction to ATP, and this newly synthesized ATP is converted to light using the luciferase/luciferin reactions. The assay was performed according to the manufacturer's instructions. Briefly, purified, recombinant ASK1 (amino acids 649-946) (10-20 ng/well) was added to a 384-well, F bottom, small volume, HIbase, white plate (Greiner Bio-One, Monroe, N.C. #784075) containing 1-2 μL 5× test compound or vehicle control (diluted 1:20 from a 100% DMSO stock solution into 1× Reaction Buffer A). The enzyme was allowed to pre-incubate with test compound from 15-120 min at 30° C. before the addition of the ATP/substrate mix (final concentrations of 50 μM ATP+250 ng MBP in 1× Reaction Buffer A). The mixture was then incubated at room temperature for 40 min before the addition of of ADP-Glo reagent and a second 40 minute room temperature incubation. Following the addition of the Kinase Detection Reagent, the plate was incubated for an additional 40 min at room temperature before reading luminescence on a FlexStation 3 (Molecular Devices, Sunnyvale, Calif.).
Measurement: IC50 < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 475.52
Compound (SMILES): CC(C)Nc1cc(Nc2nc3ccnn3cc2F)ncc1C(=O)NCCc1ccc(S(N)(=O)=O)cc1
InChIKey: BOXHZKKBKLRFSX-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 6.6 nM (pChEMBL 8.180000305175781)
Descriptors: MW 512.57, ALogP 2.45, HBA 9, HBD 4, PSA 156.4, QED 0.27
Compound (SMILES): CC(C)C(=O)NCc1ccc(F)c(-c2nc(-c3ccc(C(F)(F)F)cn3)cc(=O)[nH]2)c1Cl
InChIKey: QKWNCQMDBXPIGF-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibitory Activity Assay: Microsomes were prepared from COS-1 cells transiently transfected with a plasmid containing human mPGES-1 cDNA, and used as mPGES-1 enzyme. The mPGES-1 enzyme was diluted with a sodium phosphate buffer (pH 7.2) containing reduced glutathione (2.5 mM) and EDTA (1 mM), DMSO or a DMSO solution of a test compound (final concentration of DMSO was 1%) was added to the enzyme, and the mixture was preincubated at 4° C. for 15 minutes. Then, PGH2 as the substrate was added at a final concentration of 1 μM to start the enzymatic reaction, and after incubation at 4° C. for 4 minutes, a solution of ferric chloride (25 mM) and citric acid (50 mM) was added to terminate the enzymatic reaction. Generated PGE2 was measured by using Prostaglandin E2 Express EIA Kit (Cayman Chemical).
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 468.84, ALogP 4.58, HBA 4, HBD 2, PSA 87.74, QED 0.54
Compound (SMILES): CN1CC[C@@](O)(C#Cc2cc(-n3nc(C(N)=O)c4c3CCCC4)ccn2)C1=O
InChIKey: LPACWZWHRXPGCR-FQEVSTJZSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: NIK Enzyme Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5′-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5′-diphosphate) assay (BellBrook Labs). Purified NIK (0.5 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl2, 2 mM dithiothreitol, 10 μM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 7 μg/mLADP antibody and 5 nM ADP-MR121 633 tracer. Reactions were quenched by the addition of 20 mM 2,2′,2″,2′″-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation at 633 nm with a Fluorescence Correlation Spectroscopy Plus reader (Evotec AG). Equilibrium dissociation constant (Ki) values for NIK inhibitors are calculated from plots of activity vs. inhibitor concentration using Morrison's quadratic equation that accounts for the potential of tight binding, and by also applying the conversion factor that accounted for competitive inhibition and the concentration of substrate used in the assay relative to its Michaelis constant (Km).
Measurement: Ki = 1.19 nM (pChEMBL 8.920000076293945)
Descriptors: MW 379.42, ALogP 0.19, HBA 6, HBD 2, PSA 114.34, QED 0.72
Compound (SMILES): C[C@H](Nc1cc(N2COCC2=O)cc(F)n1)c1cc2cc(Cl)ccc2[nH]c1=O
InChIKey: OLXKFTKDYVWZBH-JTQLQIEISA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: IDH1-R132H and IDH1-R132C Enzymatic Assay: Assays were performed in a 384-well black plate. An aliquot of 250 nL of compound was incubated with 10 μL of 30 nM IDH1-R132H or 10 nM IDH1-R132C recombinant protein in assay buffer (50 mM Tris pH=7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% (w/v) Bovine Serum Albumin, and 0.01% Triton X-100) in each well at 25° C. for 15 minutes. After the plate was centrifuged briefly, an aliquot of 10 μL of 2 mM α-ketoglutarate and 20 μM NADPH solution prepared in assay buffer was then added to each well and the reaction was maintained at 25° C. for 45 minutes. An aliquot of 10 μL of diaphorase solution (0.15 U/mL diaphorase and 30 μM Resazurin in assay buffer) was added to each well. The plate was maintained at 25° C. for 15 minutes and then read on a plate reader with excitation and emission wavelengths at 535 nm and 590 nm, respectively. The IC50 of a given compound was calculated by fitting the dose response curve of inhibition of NADPH consumption at a given concentration with the four parameter logistic equation.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 402.81, ALogP 3.21, HBA 5, HBD 2, PSA 87.32, QED 0.65
Compound (SMILES): O=C(CN1CCOCC1)Nc1cc(C(=O)Nc2ccc(-c3ccccc3)cn2)ccc1OC(F)(F)F
InChIKey: OEZXPVZTYHPRBN-UHFFFAOYSA-N
Target: Protein Wnt-3a (SINGLE PROTEIN), Homo sapiens
UniProt: P56704
Assay: HCT116 TOPFlash Assay: In preparation of the assay, the two cell lines were plated 24 hours before at 10000 cells per well of a 384 micro titre plate (MTP) in 30 μL growth medium. Selective inhibitory activity for small molecules on the mutated Wnt pathway was determined after parallel incubation of both (TOP and FOP) HCT116 reporter cell lines with a compound dilution series from 50 μM to 15 nM in steps of 3.16-fold dilutions in CAFTY buffer (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl2, 5 mM NaHCO3, pH 7.4) containing 2 mM Ca2+ and 0.01% BSA. The compounds were thereby serially prediluted in 100% DMSO and thereafter in addition 50 fold into the CAFTY compound dilution buffer (described above). From this dilution 10 μL were added to the cells in 30 μL growth medium and incubated for 36 hours at 37° C. and 5% CO2. Thereafter luciferase assay buffer (1:1 mixture of luciferase substrate buffer (20 mM Tricine, 2.67 mM MgSO4, 0.1 mM EDTA, 4 mM DTT, 270 μM Coenzyme A, 470 μM Luciferin, 530 μM ATP, ph adjusted to pH 7.8 with a sufficient volume of 5M NaOH) and Triton buffer (30 mL Triton X-100, 115 mL glycerol, 308 mg Dithiothreitol, 4.45 g Na2HPO4.2H2O, 3.03 g TRIS HCl, ad 1I H2O, pH 7.8) was added as equal volume to the compound solution on the cells to determine luciferase expression as a measure of Wnt signaling activity in a luminometer.n order to determine the inhibitory activity of compounds for the WT Wnt signaling pathway, the Super TopFlash vector were cotransfected with pcDNA3 into HEK293 cells.
Measurement: IC50 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 500.48, ALogP 4.17, HBA 6, HBD 2, PSA 92.79, QED 0.51
Compound (SMILES): Cc1ncsc1C(CNC(=O)c1cc(-c2ncc(F)cn2)ccc1Cl)N1CCC(F)(F)CC1
InChIKey: MKEXAUMSPHXNHF-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Fluo-8 No Wash Calcium Assa: Extracellular binding of Bz-ATP to P2X7 receptor opens the channel and allows Ca2+ influx into the cells. This Ca2+ entry was measured in HEK-293 cells stably transfected with P2X7 receptor using Screen Quest Fluo-8 No Wash Calcium Assay Kit (AAt Bioquest, cat. 36316). Once inside the cell, the lipophilic blocking groups of Fluo-8 are cleaved by non-specific cell esterases, resulting in a negatively-charged fluorescent dye that stays inside cells. Its fluorescence increases upon binding to calcium. When HEK-293/P2X7 cells are stimulated with Bz-ATP, Ca2+ enters the cells and the fluorescence of Fluo-8 NW increases. The dye has an absorption spectrum compatible with excitation at 488 nm by argon laser sources and its emission wavelength is in the range of 515-575 nm.HEK-293 cells stably transfected with P2X7 receptor were seeded overnight in growth medium at 10,000 to 20,000 cells/well in 384-well plate. 24 hours later, the medium was removed and the cells were pre-loaded at RT for 1 hour with 20 μL/w of Fluo-8 NW. Then 10 μL/w of test compounds and reference antagonist A438079 at 3×-concentration were injected with the FLIPRTETRA and the kinetic response over a period of five minutes was monitored. A second injection of 15 μL/w of 3× reference activator (Bz-ATP at EGO was performed with the FLIPRTETRA and the signal of the emitted fluorescence was recorded for additional three minutes. All the experiment was carried out in a Low Divalent Cation Assay Buffer (0.3 mM Ca2+ and 0 mM Mg2+). The effect of the test compounds was measured as percent inhibition vs the reference antagonist and IC50 values were calculated accordingly.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 495.96, ALogP 4.9, HBA 6, HBD 1, PSA 71.01, QED 0.53
Compound (SMILES): CO[C@H]1C[C@H](NC(=O)Nc2cc3cc(-c4cnc5c(c4C)NCCO5)c(F)c(N)c3cn2)C1
InChIKey: YOKQTLYYNFAINQ-HDJSIYSDSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: TBD: TBD
Measurement: Ki < 0.013 nM (pChEMBL 10.88599967956543)
Descriptors: MW 452.49, ALogP 3.43, HBA 7, HBD 4, PSA 123.42, QED 0.45
Compound (SMILES): Cn1ncc2c1CN(C1CCOCC1)c1nccc3[nH]nc-2c13
InChIKey: SOAIDXSOSFIZBG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK2 Assay: JAK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.05% 0-mercaptoethanol, 1 mg/ml BSA) is assayed against PDKtide (KTFCGTPEYLAPEVRREPRILSEEEQ-EMFRDFDYIADWC) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.05% 0-mercaptoethanol, 100 μM substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature Assays are stopped by addition of 5 μl of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.
Measurement: IC50 = 920 nM (pChEMBL 6.039999961853027)
Descriptors: MW 310.36, ALogP 1.86, HBA 6, HBD 1, PSA 71.86, QED 0.74
Compound (SMILES): C[C@@H](Nc1nc(-c2noc(=O)[nH]2)nc2nc(N3CCOC[C@H]3c3ccccc3)n(Cc3ccc(Cl)c(F)c3)c12)C1CCC1
InChIKey: WNTSSWMQMGJUBQ-HXOBKFHXSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 0.8475 nM (pChEMBL 9.069999694824219)
Descriptors: MW 605.07, ALogP 5.19, HBA 10, HBD 2, PSA 126.99, QED 0.25
Compound (SMILES): Cc1nc2c(C3CC3)cc(C#Cc3nc(N4CCCC4)nn3C)nn2c1C
InChIKey: PQWUHXREIWGRJB-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Inhibition Assay: The human PDE10A full length assay was performed in 96-well micro titer plates. The reaction mixture of 50 μl contained 20 mM HEPES pH=7.5/10 mM MgCl2/0.05 mg/ml BSA (Sigma cat. #A-7906), 50 nM cGMP (Sigma, cat. #G6129) and 50 nM [3H]-cGMP (GE Healthcare, cat. #TRK392 S.A. 13.2 Ci/mmol), 3.75 ng/well PDE10A enzyme (Enzo Life Science, Lausen, Switzerland cat #SE-534) with or without a specific test compound. A range of concentrations of the potential inhibitor was used to generate data for calculating the concentration of inhibitor resulting in 50% of the effect (e.g. IC50, the concentration of the competitor inhibiting PDE10A activity by 50%). Non-specific activity was tested without the enzyme. The reaction was initiated by the addition of the substrate solution (cGMP and [3H]-cGMP) and allowed to progress for 20 minutes at room temperature. The reaction was terminated by adding 25 μl of YSi-SPA scintillation beads (GE Healthcare, cat. #RPNQ0150) in 18 mM zinc sulphate solution (stop reagent). After 1 h under shaking, the plate was centrifuged one minute at 170 g to allow beads to settle. Afterwards, radioactive counts were measured on a Perkin Elmer TopCount Scintillation plate reader.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 361.45, ALogP 2.35, HBA 7, HBD 0, PSA 64.14, QED 0.66
Compound (SMILES): C[C@H](CC(=O)N1CCC(C(=O)c2cnn(C)c2)CC1)Cc1nc2ccccc2c(=O)[nH]1
InChIKey: QXYNHQXOVDACBV-HNNXBMFYSA-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 = 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 421.5, ALogP 2.35, HBA 6, HBD 1, PSA 100.95, QED 0.62
Compound (SMILES): CCOC(=O)c1nn[nH]c1Oc1cccc(C#CCC(C)(C)C)c1
InChIKey: QMCUYTFLVXCKBZ-UHFFFAOYSA-N
Target: 2-Hydroxyacid oxidase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UJM8
Assay: In-Vitro Human Glycolate Oxidase (hGOX) Assay: The in-vitro glycolate oxidase assay was performed using recombinant full-length human hydroxyacid oxidase 1 (HAO1), the equivalents of hGOX. The enzyme was obtained from AbCam (Catalog #ab113144) and was purified using conventional chromatography to >95% purity. Purified HAO1 was dissolved in assay buffer consisting of 10 mM NaCl, 110 mM KCl, 2 mM MgCl2, 50 mM HEPES (pH 7.4), and 0.01% Triton X-100. The assay used Corning 3575 384-well flat bottom, low flange, non-binding surface, black polystyrene plates.Test compounds in DMSO were preincubated at different concentrations with purified recombinant human GO (6 nM) for 10 min, followed by the addition of glycolate substrate (85 μM) to start the reaction. The plates were incubated for 10 min at room temperature, at which point Amplex red reagent (50 μM) was added.The fluorescence intensity signal was measured on a VariosKan LUX instrument using an excitation of 560 nm and an emission of 590 nm. The IC50 values were calculated using Graphpad Prism.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 327.38, ALogP 3.56, HBA 5, HBD 1, PSA 77.1, QED 0.69
Compound (SMILES): Cc1ccc([C@@H](NC(=O)c2ccc(C#N)c(C)c2)c2ccc(Cl)c(F)c2)nc1
InChIKey: FDLLRRWCGFUPBC-NRFANRHFSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: TBD: TBD
Measurement: IC50 = 3300 nM (pChEMBL 5.480000019073486)
Descriptors: MW 393.85, ALogP 4.88, HBA 3, HBD 1, PSA 65.78, QED 0.69
Compound (SMILES): CCn1c(-c2cnc(C)nc2)nc2c(O[C@H]3CCN(C(=O)[C@@H](N)C(C)C)C3)ncnc21
InChIKey: NXLBIAAHKPNXPP-HOTGVXAUSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Biochemical Assay: The PI3-Kinase biochemical assay was optimized using the HTRF kit provided by Upstate (Millipore). The assay kit contains six reagents: 1) 4× Reaction Buffer; 2) native PIP2 (substrate); 3) Stop (EDTA); 4) Detection Mix A (Streptavidin-APC); 5) Detection Mix B (Eu-labeled Anti-GST plus GST-tagged PH-domain); 6) Detection Mix C. In addition, the following items were obtained or purchased; PI3Kinase (alpha 14-602, beta 14-603, gamma 14-558 and delta 14-604 from Upstate; Millipore), dithiothreitol (Sigma, D-5545), Adenosine-5′ triphosphate (InVitrogen, Cat#AS001A), native PIP3 (PI(3,4,5)P3, diC8, H+, CELLSIGNALS, INC. Cat #907) DMSO (Sigma, 472301).PI3Kinase Reaction Buffer is prepared by dilution the stock 1:4 with de-ionized water. DTT, PIP2 and Biotin-PIP3 were added to 1536 assay plate at a final concentration of 5 mM, 5 mM and 25 nM on the day of use. Enzyme addition and compound pre-incubation are initiated by the addition of 1.25 ul of PI3K (at twice its final concentration) in the 1× reaction buffer to all wells using a BioRaptor. Plates are incubated at room temperature for 15 minutes. Reactions are initiated by addition of 1.25 ul of 2× substrate solution (PIP2 and ATP in 1× reaction buffer) using BioRaptor. Plates are incubated in humidified chamber at room temperature for one hour. Reactions are quenched by addition of 0.625 uL of stop solution to all wells using the BioRaptor. The quenched reactions are then processed to detect product formation by adding 0.625 uL of Detection Solution to all wells using the BioRaptor (Detection mix C, Detection Mix A, and Detection Mix B combined together in an 18:1:1 ratio prepared 2 hours prior to use). Following a one hour incubation in the dark, the HTRF signal is measured on the Envision plate reader set for 330 nm excitation and dual emission detection at 620 nM (Eu) and 665 nM (APC).
Measurement: IC50 = 9.8 nM (pChEMBL 8.010000228881836)
Descriptors: MW 424.51, ALogP 1.57, HBA 9, HBD 1, PSA 124.94, QED 0.63
Compound (SMILES): Nc1ccccc1NC(=O)c1ccc2c(cnn2Cc2cccnc2)c1
InChIKey: VQWRDIFVQPSZFZ-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Enzyme Inhibition: The HDAC activity inhibition assay was performed as follows to determine the ability of a test compound to inhibit HDAC enzymatic activity. Serial dilutions of HDAC inhibitors were prepared in HDAC assay buffer (25 mM Tris/HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, pH 8) in 96-well assay plates (Fisher scientific, #07-200-309) and were pre-incubated for 2 hours at room temperature in the presence of 125 μg/ml BSA and purified HDAC1 (BPS Bioscience, San Diego, Calif., #50051), HDAC2 (BPS Bioscience, #50053), or HDAC3/NcoR2 (BPS Bioscience, #50003) at concentrations of 1.25, 1.32, and 0.167 μg/mL, respectively. Following pre-incubation, Fluor-de-Ly substrate (Enzo Life Sciences, Plymouth Meeting, Pa., BML-KI104-0050) was added to a final concentration of 10 μM and plates were further incubated for 30 minutes at room temperature. The enzymatic reaction was stopped by addition of Trichostatin A (Sigma-Aldrich, St Louis, Mo., #T8552, final concentration: 100 nM) and trypsin (MP Biomedicals, Solon, Ohio, #02101179) was added to reach a final concentration of 100 μg/mL. After a 15 minute incubation at room temperature, fluorescence was recorded using a Spectramax M2 fluorometer (Molecular Devices, Sunnyvale, Calif.) with excitation at 365 nm and emission at 460 nm.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 343.39, ALogP 3.31, HBA 5, HBD 2, PSA 85.83, QED 0.56
Compound (SMILES): CNS(=O)(=O)CCCn1c(CN2C(=O)C3(CC3)c3ccncc32)cc2cc(Cl)cnc21
InChIKey: YNTFQXBCCZOWCP-UHFFFAOYSA-N
Target: Protein M2-1 (SINGLE PROTEIN), Human respiratory syncytial virus A (strain A2)
UniProt: P04545
Assay: Viral Cytopathic Effect (CPE) Assay: To measure anti-RSV activity of compounds, 96-well plates are seeded with 6×103 cells per well in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). Cells are infected the next day with sufficient RSV Long strain (ATCC) to produce an approximately 80-90% cytopathic effect after 6 days, in the presence of serial half-log diluted compound in a total volume of 200 μA per well. The viability of cells is assessed after 6 days using Cell Counting kit-8 (Dojindo Molecular Technologies). The absorbance at 450 nm and referenced at 630 nm is measured to determine 50% effective concentration (EC50)The compounds of the present invention were tested for their anti-RSV activity, and the activation as described herein. The Examples were tested in the above assay and found to have EC50 of about 0.0001 μM to about 10 μM. Particular compound of formula (I) were found to have EC50 of about 0.0001 μM to about 1 μM. Further particular compound of formula (I) were found to have EC50 of about 0.0001 μM to about 0.1 μM.
Measurement: EC50 = 57 nM (pChEMBL 7.239999771118164)
Descriptors: MW 459.96, ALogP 2.6, HBA 6, HBD 1, PSA 97.19, QED 0.59
Compound (SMILES): COc1ncc(-c2cc(CNCCO)c3c(N)ncnn23)cc1C(=O)Nc1cnn(Cc2ccccc2)c1
InChIKey: FGHRPENBSMMNAC-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 2500 nM (pChEMBL 5.599999904632568)
Descriptors: MW 513.56, ALogP 1.96, HBA 11, HBD 4, PSA 157.51, QED 0.2
Compound (SMILES): CC(C)n1c(-c2cnoc2)nc2c1C(c1ccc(Cl)cc1)N(c1ccc(=O)n(C)c1)C2=O
InChIKey: DNXFUDZUKZRLEU-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: In-Vitro Binding Assay: All assays were performed in 384 well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Perkin Elmer/Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO HummingBird nanodispenser (Zinsser Analytic). The assay was started by stepwise addition of 4.5 ul per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 45 nM His-Brd2(60-472) or 45 nM His-Brd3(20-477) or 45 nM His-Brd4(44-477) all proteins produced in-house) and 4.5ul per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 60 nM acetyl-histone H4 (AcK 5, 8, 12, 16) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 ul per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 3 nM Eu-labeled anti-His6 antibody, 21 nM streptavidin-allophycocyanin) were added. After 35 minutes incubation at 30° C., plates were measured in a Perkin Elmer EnVision multilabel reader. Concentrations causing 50% inhibition (IC50 values) were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.
Measurement: IC50 = 220 nM (pChEMBL 6.659999847412109)
Descriptors: MW 449.9, ALogP 4.22, HBA 7, HBD 0, PSA 86.16, QED 0.47
Compound (SMILES): COCCNC1CCC(c2nc3c(C(C)C)c(-c4cc(OC)c5ncnn5c4)[nH]c3cc2C)CC1
InChIKey: UOFHUHFRAORPOM-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 > 3125 nM (pChEMBL 5.505000114440918)
Descriptors: MW 476.63, ALogP 4.98, HBA 7, HBD 2, PSA 89.36, QED 0.35
Compound (SMILES): O=C(NCC(c1scnc1C(F)F)N1CCC(OCc2cc(F)cc(F)c2)CC1)c1c(F)cccc1Cl
InChIKey: PKXYTHTWRYGTJM-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: Screen Quest Fluo-8 No Wash Calcium Assay Kit: Ca2+ influx was measured in HEK-293 cells stably transfected with the receptor using Screen Quest Fluo-8 No Wash Calcium Assay Kit (AAt Bioquest®). Briefly, once inside the cells, the lipophilic blocking groups of Fluo-8 are cleaved by non-specific cell esterases, resulting in a negatively-charged fluorescent-dye that stays inside cells. Its fluorescence increases upon binding to calcium. When HEK-293/P2X7 cells were stimulated with BzATP, Ca2+ entered the cells and the fluorescence of Fluo-8 NW increaseed. The dye absorption spectrum was compatible with excitation at 488 nm by argon laser sources and its emission wavelength was in the range of 515-575 nm.To routinely test the compounds, HEK-293 cells stably transfected with rat P2X7R were seeded overnight in growth medium at 10000, 15000 or 20000 cells/well in 384-well plate, according to the level of response after thawing. 24 hours later, the medium was removed and the cells were pre-loaded at RT for 1 hour with 20 μL/w of Fluo-8 NW prepared in Tyrode 0.3 mM Ca2+/Mg2+-free. Compounds of the invention were tested at 8 concentrations (4 replicates for each concentration): 10-3.16-1-0.316-0.1-0.0316-0.01 and 0.00316 μM, in the same plate.Compounds were tested at FLIPRTETRA according to the following method:    first injection at FLIPRTETRA of 10 μL of 3× test compound (in Tyrode's buffer 0.3 mM Ca2+/Mg2+-free+DMSO 0.5% final concentration)    5′ incubation    second injection at FLIPRTETRA of 15 □L of 3× BzATP at ECK) (in Tyrode's buffer 0.3 mM Ca2+/Mg2+-free+BSA 0.0003% final concentration)    Fluorescence recording for 3′Between one plate and the following, tips were extensively washed with water, then with 100% DMSO and finally with water to avoid carry-over inside the tips.The effect of the test compounds was measured as percent inhibition vs a reference antagonist and IC50 values were calculated accordingly.
Measurement: IC50 = 578 nM (pChEMBL 6.239999771118164)
Descriptors: MW 559.99, ALogP 6.3, HBA 5, HBD 1, PSA 54.46, QED 0.31
Compound (SMILES): Cc1cc(N2CCOCC2)nc(C)c1-c1nc2ccncc2n1-c1ccc(F)cc1
InChIKey: YCDZUPIXZOHNMV-UHFFFAOYSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: Biological Assay: SSAO: All primary assays were performed at RT. with purified recombinantly expressed human SSAO. Enzyme was prepared essentially as described in Öhman et al. (Protein Expression and Purification 46 (2006) 321-331). In addition, secondary- and selectivity assays were performed using SSAO prepared from various tissues or purified rat recombinant SSAO. The enzyme activity was assayed with benzylamine as substrate by measuring either benzaldehyde production, using 14C-labeled substrate, or by utilizing the production of hydrogen peroxide in a horseradish peroxidase (HRP) coupled reaction. Briefly, test compounds were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM. Dose-response measurements were assayed by either creating 1:10 serial dilutions in DMSO to produce a 7 point curve or by making 1:3 serial dilutions in DMSO to produce 11 point curves. The top concentrations were adjusted depending on the potency of the compounds and subsequent dilution in reaction buffer yielded a final DMSO concentration≤2%.
Measurement: IC50 = 700 nM (pChEMBL 6.159999847412109)
Descriptors: MW 403.46, ALogP 4.08, HBA 6, HBD 0, PSA 56.07, QED 0.52
Compound (SMILES): O=C(CN1CCN(c2cccc3c2cnn3-c2ccccc2F)C1=O)C1CCC1
InChIKey: QMQKOGIFYMTZJD-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FLIPR Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 μg/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 μL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 μM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 μL assay buffer containing 2.5 μM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR®TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics.
Measurement: IC50 = 635 nM (pChEMBL 6.199999809265137)
Descriptors: MW 392.43, ALogP 3.78, HBA 4, HBD 0, PSA 58.44, QED 0.66
Compound (SMILES): Cc1nc2cc([C@@H]3CCCCN3c3ncnc4cccc(C)c34)nn2c2c1CCCC2
InChIKey: RGRACESGLXICBB-QFIPXVFZSA-N
Target: Protein M2-1 (SINGLE PROTEIN), Human respiratory syncytial virus A (strain A2)
UniProt: P04545
Assay: Antiviral Activity: Black 384-well clear-bottom microtiter plates (Corning, Amsterdam, The Netherlands) were filled via acoustic drop ejection using the echo liquid handler (Labcyte, Sunnyvale, Calif.). 200 nL of compound stock solutions (100% DMSO) were transferred to the assay plates. 9 serial 4-fold dilutions of compound were made, creating per quadrant the same compound concentration. The assay was initiated by adding 10 μL of culture medium to each well (RPMI medium without phenol red, 10% FBS-heat inactivated, 0.04% gentamycin (50 mg/mL). All addition steps are done by using a multidrop dispenser (Thermo Scientific, Erembodegem, Belgium). Next, rgRSV224 virus (MOI=1) diluted in culture medium was added to the plates. rgRSV224 virus is an engineered virus that includes an additional GFP gene (Hallak L K, Spillmann D, Collins P L, Peeples M E. Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection; Journal of virology (2000), 74(22), 10508-13) and was in-licensed from the NIH (Bethesda, Md., USA). Finally, 20 μL of a HeLa cell suspension (3,000 cells/well) were plated. Medium, virus- and mock-infected controls were included in each test. The wells contain 0.05% DMSO per volume. Cells were incubated at 37° C. in a 5% CO2 atmosphere. Three days post-virus exposure, viral replication was quantified by measuring GFP expression in the cells by an in house developed MSM laser microscope (Tibotec, Beerse, Belgium). The EC50 was defined as the 50% inhibitory concentration for GFP expression. In parallel, compounds were incubated for three days in a set of white 384-well microtiter plates (Corning) and the cytotoxicity of compounds in HeLa cells was determined by measuring the ATP content of the cells using the ATPlite kit (Perkin Elmer, Zaventem, Belgium) according to the manufacturer's instructions. The CC50 was defined as the 50% concentration for cytotoxicity.
Measurement: EC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 412.54, ALogP 4.9, HBA 6, HBD 0, PSA 59.21, QED 0.47
Compound (SMILES): Cc1cnc(Oc2ccc(/N=c3\[nH]c(=O)n(CC(O)CO)c(=O)n3Cc3ccc(Cl)cc3)cc2)s1
InChIKey: GXTUUXBYFHJEEA-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 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 515.98, ALogP 2.18, HBA 10, HBD 3, PSA 134.73, QED 0.33
Compound (SMILES): Cc1cc(C2CCCN(Cc3ccc(CC(C)C)cc3)C2)n2ncnc2n1
InChIKey: SJHAMDBCXZVNSZ-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 363.51, ALogP 4.01, HBA 5, HBD 0, PSA 46.32, QED 0.69
Compound (SMILES): O=c1[nH]nc2c3c(cccc13)N=C1CNCCN12
InChIKey: MDCGHJKIHMHTTA-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Universal PARP colorimetric assay kit: US Trevigen company
Measurement: IC50 = 90.5 nM (pChEMBL 7.039999961853027)
Descriptors: MW 241.25, ALogP 0.38, HBA 5, HBD 2, PSA 73.38, QED 0.7
Compound (SMILES): CC1CCn2ncc(Nc3nccc(-c4cc(C#N)c5c(c4)[C@@](C)(CO)CN5)n3)c2O1
InChIKey: CTILFWYUBMVGBP-VOVKCACBSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition of Auto-Phosphorylation of Recombinant Human NF-kappaB-Inducing Kinase (NIK1/AP3K14) Activity: NIK/MAP3K14 auto-phosphorylation activity was measured using the AlphaScreen® (αscreen) format (Perkin Elmer). All compounds tested were dissolved in dimethyl sulfoxide (DMSO) and further dilutions were made in assay buffer. Final DMSO concentration was 100 (v/v) in assays. Assay buffer was 50 mM Tris pH 7.5 containing 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (dithiothreitol), 0.1 mM Na3VO4, 5 mM MgCl2, 0.01% Tween® 20. Assays were carried out in 384 well Alphaplates (Perkin Elmer). Incubations consisted of compound, 25 microM Adenosine-5′-triphosphate (ATP), and 0.2 nM NIK/MAP3K14. Incubations were initiated by addition of GST-tagged NIK/MAP3K14 enzyme, carried out for 1 h at 25° C. and terminated by addition of stop buffer containing anti-phospho-KK Ser176/180 antibody. Protein A Acceptor and Glutathione-Donor beads were added before reading using an EnVision® Multilabel Plate Reader (Perkin Elmer). Signal obtained in the wells containing blank samples was subtracted from all other wells and IC50's were determined by fitting a sigmoidal curve to % inhibition of control versus Log10 compound concentration.
Measurement: IC50 = 5.9 nM (pChEMBL 8.229999542236328)
Descriptors: MW 417.47, ALogP 2.8, HBA 9, HBD 3, PSA 120.91, QED 0.59
Compound (SMILES): Cn1cnc(S(=O)(=O)N2C[C@@H](Nc3cccc(F)c3)[C@H](c3ncccc3F)C2)c1
InChIKey: GLXKQKFEMKPLMO-NVXWUHKLSA-N
Target: Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P26572
Assay: Radioligand Binding Assay: Radioligand binding assays using recombinant CHO cell membranes expressing human GlyT1: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 1000 nM (pChEMBL 6)
Descriptors: MW 419.46, ALogP 2.36, HBA 6, HBD 1, PSA 80.12, QED 0.69
Compound (SMILES): CN(C)C(=O)CN(CCCCc1ccc2c(n1)NCCC2)CC[C@H](Nc1ncnc2c1cnn2C)C(=O)O
InChIKey: WRFIXPUWVYCVEA-NRFANRHFSA-N
Target: Integrin alpha-5/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P18564
Assay: Binding Assay: Microplates were coated with recombinant human integrin αVβ6 (2 μg/mL) in PBS (100 μL/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). The plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 523.64, ALogP 1.78, HBA 10, HBD 3, PSA 141.4, QED 0.28
Compound (SMILES): CNC(=O)c1cc(Nc2nccc(-c3cc(C#N)c4c(c3)[C@@](C)(CO)CN4)n2)c(=O)n2c1CCC2
InChIKey: SNJKGIPRAINBTI-RUZDIDTESA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition of Auto-Phosphorylation of Recombinant Human NF-kappaB-Inducing Kinase (NIK1/AP3K14) Activity: NIK/MAP3K14 auto-phosphorylation activity was measured using the AlphaScreen® (αscreen) format (Perkin Elmer). All compounds tested were dissolved in dimethyl sulfoxide (DMSO) and further dilutions were made in assay buffer. Final DMSO concentration was 100 (v/v) in assays. Assay buffer was 50 mM Tris pH 7.5 containing 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (dithiothreitol), 0.1 mM Na3VO4, 5 mM MgCl2, 0.01% Tween® 20. Assays were carried out in 384 well Alphaplates (Perkin Elmer). Incubations consisted of compound, 25 microM Adenosine-5′-triphosphate (ATP), and 0.2 nM NIK/MAP3K14. Incubations were initiated by addition of GST-tagged NIK/MAP3K14 enzyme, carried out for 1 h at 25° C. and terminated by addition of stop buffer containing anti-phospho-KK Ser176/180 antibody. Protein A Acceptor and Glutathione-Donor beads were added before reading using an EnVision® Multilabel Plate Reader (Perkin Elmer). Signal obtained in the wells containing blank samples was subtracted from all other wells and IC50's were determined by fitting a sigmoidal curve to % inhibition of control versus Log10 compound concentration.
Measurement: IC50 = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 471.52, ALogP 1.9, HBA 9, HBD 4, PSA 144.96, QED 0.44