lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cccc(-c2nc3nc(C(=O)O)nc(NC4CCCC(C)C4)c3n2Cc2ccc(C(F)(F)F)cc2)c1
InChIKey: KDRQBSPYKXRAHN-UHFFFAOYSA-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 = 566.8 nM (pChEMBL 6.25)
Descriptors: MW 523.56, ALogP 6.56, HBA 6, HBD 2, PSA 92.93, QED 0.3
Compound (SMILES): Cc1c(-c2ccccc2)nc2ccc(Br)cc2c1C(=O)N[C@H](C)[C@@H](CC(=O)O)c1ccccc1Cl
InChIKey: LHJGLUPCRUHDLL-DYESRHJHSA-N
Target: Prostaglandin F2-alpha receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43088
Assay: In Vitro Test of Inhibition of Human FP Receptor Activity: For the characterization of test substances in respect of FP antagonism, PGF2α-induced calcium flux in FP-expressing CHEM1 cells (Millipore, HTS093C) was used.3000 cells in 30 μl of full medium [DMEM F12, 10% FCS, 1.35 mM sodium pyruvate, 20 mM HEPES, 4 mM GlutaMAX, 2% sodium bicarbonate, 1% Pen/Strep, 1% 100× non-essential amino acids] are sown per well of a 384 multititre plate (from Greiner, TC plate, black with clear base) and incubated at 33° C., 5% CO2 for 24 hours. Prior to the measurement, the medium is replaced by 30 μl of Fluo-8 AM loading buffer [calcium-free Tyrode (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl2, 4.8 mM NaHCO3, pH 7.4), 2 mM CaCl2, 6.3 mM Probenecid, 5 μM Fluo-8 AM, 0.0112% Pluronic®] and incubated at 37° C., 5% CO2 for 30 minutes. The test substance is prepared in DMSO in various concentrations as a dose-response curve (starting concentration 10 mM, dilution factor 3.16) and prediluted 1:50 with calcium-free Tyrode, 2 mM CaCl2, 0.002% SmartBlock (from CANDOR Bioscience GmbH). 10 μl of the prediluted substance solution are added to the Fluo-8-laden cells and incubated at 37° C., 5% CO2 for 10 minutes. The FP receptor is activated by adding 40 μl of 2 nM (final concentration) PGF2α in calcium-free Tyrode, 2 mM CaCl2, 0.002% SmartBlock, and the calcium flux is determined by measuring the fluorescence at ex. 470 nm/em. 525 nm in a fluorescence measuring instrument (FLIPR Tetra®, Molecular Devices) for 120 seconds.
Measurement: IC50 = 960 nM (pChEMBL 6.019999980926514)
Descriptors: MW 551.87, ALogP 7, HBA 3, HBD 2, PSA 79.29, QED 0.26
Compound (SMILES): Cn1cc(-c2cc3[nH]cnc3cc2Oc2ccc(F)cc2F)c2cn[nH]c2c1=O
InChIKey: QPHFLUZYEBTKLI-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4 AlphaScreen Assay: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 20 μL for BD1 and 40 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature for 75 min. in the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% CHAPS, 0.01% BSA), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4), 3.8 nM (BRD4-BD1, BPS Bioscience #31040) or 20 nM (BRD4-BD2, BPS Bioscience #31041). The reaction followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at 4 g/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech). IC50 determination was performed by fitting the curve of percent control activity versus the log of the inhibitor concentration using the GraphPad Prism 5.0 software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 393.35, ALogP 3.88, HBA 5, HBD 2, PSA 88.59, QED 0.49
Compound (SMILES): COC[C@H]1CCCN1C(=O)c1cccc(Nc2nc(N3CCC[C@H](N)C3)ncc2C(N)=O)c1
InChIKey: ZAFAORJDJYRHQM-FUHWJXTLSA-N
Target: Calcium/calmodulin-dependent protein kinase type 1D (SINGLE PROTEIN), Homo sapiens
UniProt: Q8IU85
Assay: Biochemical CaMK1D Enzymatic Activity Assay: The following describes an ADP-Glo Kinase Assay, which measures the ADP formed from a kinase reaction; the ADP generated is converted into ATP and is used to generate light in a luciferase reaction. The assay is used to assess the effect of compounds on the activity of purified CaMK1 D.Materials and Solutions:All reagents are from Sigma-Aldrich unless otherwise specified. ADP-Glo Kinase Assay (Promega, V9102). His-tagged CaMK1D_1-385 (Fisher Scientific, PR6770A). Autocamtide-2 (SignalChem, A15-58). Calmodulin (Merck, 208694). 1M Tris-HCl pH7.5 (Fisher Scientific, 10123722). 1M DTT (Fisher Scientific, 10674545). Calcium chloride (C1016). Magnesium Chloride (M8266). DMSO (D8418). Autocamtide-2 provided as a lyophilised powder and prepared as a 10 mM stock in MilliQ water. RB: 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1 CaCl2), just prior to use 1M DTT was added to a final concentration of 2 mM.Assay Protocol: 7.88 μL reaction mixture (including: calmodulin, Autocamtide-2, CaMK1 D in RB) was incubated with 12 μL test compound in 100% DMSO. To start the reaction 4 μL of ATP mixture were added. Final assay concentrations: 3 nM CaMK1D, 1 μM Calmodulin, 125 μM Autocamtide-2 and 10 μM ATP. Plates were incubated at 25C for 2 hours prior to the 1:1 addition of ADP-Glo reagent. Plates were incubated for a further 1 hour prior to the 1:1 addition of ADP-Glo substrate. After 30 minutes plates read with the EnVision® Multilabel Plate Reader, using Luminescence 700. Compound IC50 was determined using a 4-parameter equation and are reported in nM.
Measurement: IC50 = 414 nM (pChEMBL 6.380000114440918)
Descriptors: MW 453.55, ALogP 1.5, HBA 8, HBD 3, PSA 139.7, QED 0.57
Compound (SMILES): CC1c2ccc(C#N)cc2CCN1C(=O)CN1Cc2ccc(-c3nc(NC4CCOCC4)ncc3Cl)cc2C1=O
InChIKey: DGFNHYCMWDUSIP-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 In Vitro Inhibition Assay: Activity of ERK2 enzyme (Life Technologies) was determined using a time-resolved fluorescence format measuring the phosphorylation of a truncated version of Activating transcription factor 2 labelled with green fluorescent protein (ATF2-GFP) (Life Technologies). Assay reactions containing 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 mM ATF2-GFP, 20 μM ATP and 0.25 nM ERK2 were set up in the presence of compound and allowed to proceed for 30 min at room temperature. Reactions were then stopped using TR-FRET dilution buffer (Life Technologies), 25 mM EDTA and 2 nM Tb-Anti-pATF2 (Thr71) (Life Technologies). After a further incubation period of at least 30 minutes, fluorescence was read on a Pherastar reader (Lanthascreen optic module; excitation 340 nm, emission 520 nm (channel A), 495 nm (channel B)). The ratio between A and B counts was used to calculate signal. IC50 values were calculated using a sigmoidal dose response equation (Prism GraphPad software, La Jolla, Calif., USA).
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 557.05, ALogP 4.36, HBA 7, HBD 1, PSA 111.45, QED 0.5
Compound (SMILES): CC1(C)[C@H](c2nc(-c3ccc([C@@](C)(O)c4cccc(C(F)(F)F)c4)cc3)c3c(N)nccn23)CC[C@]1(C)C(=O)O
InChIKey: YOQBUNDUQZOQJR-HUXLMHJOSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity Assay: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 μM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 μL) was dispensed, followed by the addition of 7.5 μL of 1× kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/μL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 μL 1× kinase buffer containing 8 μM biotinylated A5 peptide (Biotin-EQEDEPEGDYFEWLE-NH2) (SEQ.ID.NO.: 1), and 100 μM ATP. The final reaction in each well of 10 μL consists of 50 pM hBTK, 2 μM biotin-A5-peptide, and 25 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1× quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10) compound concentrations.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 552.6, ALogP 6.25, HBA 6, HBD 3, PSA 113.74, QED 0.27
Compound (SMILES): Cc1nc(N[C@H](C)c2cccc(C(F)F)c2F)c2cc(C3=CCN(C4COC4)CC3)c(=O)n(C)c2n1
InChIKey: POLHTSVPKPRUQH-CQSZACIVSA-N
Target: Son of sevenless homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07889
Assay: Inhibition of SOS1 Nucleotide Exchange Activity: The purpose of this assay was to characterize the inhibitory activity of compounds on SOS1 nucleotide exchange of KRAS. Data was reported as IC50 values based on the TR-FRET signal.Notethe Following Protocol Describes a Procedure for Monitoring the Inhibition of SOS1 Nucleotide Exchange Activity of Wild-Type KRAS in Response to a Compound of the Invention. Other KRAS Mutants and RAS Isoforms May be Employed.In assay buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 0.05% Tween-20, 0.1% BSA, 1 mM DTT, concentration series of test compounds were generated spanning 100 μM to 1.7 nM over eleven 3-fold serial dilutions in a 384-well assay plate at a volume of 20 μL. The purified tagless catalytic domain of SOS1 (residues 564-1049) was first diluted in assay buffer at a concentration of 100 nM, and then 20 μL of the SOS1 containing solution was directly dispensed into compound plates. The SOS1/compound mixture was incubated at room temperature with constant mixing on an orbital shaker for 20 minutes to allow the reaction to reach equilibrium. A KRAS mixture was prepared by diluting 66.7 nM avi-tagged KRAS (residue 1-169), 3.33 nM Streptavidin-Tb and 333 nM EDA-GTP-DY-647P1 in assay buffer. This mixture was prepared immediately before addition to the SOS1/compound mixture to prevent intrinsic nucleotide exchange. Then 5 μL of the pre-incubated SOS1/compound mixture and 7.5 μL of the KRAS mixture were added sequentially in a 384-well low volume black round bottom plate and incubated at room temperature with constant shaking for 30 minutes. Time-resolved fluorescence was measured on a PerkinElmer Envision plate reader. DMSO and 10 μM of compound (i) were used as negative and positive controls, respectively.
Measurement: Kd = 61 nM (pChEMBL 7.210000038146973)
Descriptors: MW 499.54, ALogP 4.37, HBA 7, HBD 1, PSA 72.28, QED 0.54
Compound (SMILES): CC(C)(O)c1cc2cc(Cl)ccc2n1S(=O)(=O)c1cc(Br)cc(Cl)c1O
InChIKey: APVTXDUNTBOOLM-UHFFFAOYSA-N
Target: ATP-citrate synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P53396
Assay: ADP-Glo Assay: Compounds of the present invention were evaluated in an ADP-GLO assay as follows:a) Dilute cpd 1:3 in succession in DMSO by hand for each cpds for 12 ptsb) Add 0.1 μL diluted cpd solution to assay plate, each dose with 2 replicatesc) Centrifuge 1000 RPM for 1 mind) Add 5 μL ACL working solution to 384-well assay plate, centrifuge 1000 RPM for 1 mine) Incubate at 25° C. for 15 minf) Add 5 μL substrate working solution to initiate reactiong) Final ACL reaction concentrations: 3 nM ACL, 15 μM ATP, 3 μM CoA, 300 μM Citrate, 0.01% Brij35, 4 mM DTT, 1% DMSO;h) Reference final conc: 30 uM starting Conc, 3× dilution, 11+0 points. Test cpd conc: 30/100 uM starting Conc, 3× dilution, 11+0 points.i) Incubate at 25° C. for 60 minj) Add 10 μL ADP Glo reagent, centrifuge 1000 RPM for 1 mink) Incubate at 25° C. for 40 minl) Add 20 μL kinase detection reagent, centrifuge 1000 RPM for 1 minm) Incubate at 25° C. for 40 minn) Read on Envision for US LUM as RLU.
Measurement: IC50 = 10250 nM (pChEMBL 4.989999771118164)
Descriptors: MW 479.18, ALogP 4.88, HBA 5, HBD 2, PSA 79.53, QED 0.56
Compound (SMILES): N#C[C@H]1CC[C@H](C(=O)N2Cc3cccnc3Nc3ccc(N4CCOCC4)cc32)CC1
InChIKey: OUIDMPDLMIQUES-IYARVYRRSA-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 = 801 nM (pChEMBL 6.099999904632568)
Descriptors: MW 417.51, ALogP 3.84, HBA 6, HBD 1, PSA 81.49, QED 0.8
Compound (SMILES): COc1cnc2c(Nc3ccc(F)c([C@]45CCC[C@H]4S(=O)(=O)N(C)C(=N)N5)c3)nccc2n1
InChIKey: XPAQUQVFMRPMDY-IIBYNOLFSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE-1 Ki Assay (BACE-1 HTRF FRET Assay): The compounds of the invention were assessed for their ability to inhibit BACE-1 using the following assay. The resulting values are reported in the tables above.The following reagents were used in this assay. Na+-Acetate pH 5.0; 1% Brij-35; Glycerol; Dimethyl Sulfoxide (DMSO); Recombinant human soluble BACE-1 catalytic domain (>95% pure); APP Swedish mutant peptide substrate (QSY7-APPswe-Eu): QSY7-EISEVNLDAEFC-Europium-amide.A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE-1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE-1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE-1 enzyme. Inhibition of BACE-1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 ul are preincubated with purified human BACE-1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 μl in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 milisecond delay followed by a 400 millisecond acquisition time window. Inhibitor IC50 values are derived from non-linear regression analysis of concentration response curves. Ki values are then calculated from IC50 values using the Cheng-Prusoff equation using a previously determined μm value of 8 μM for the QSY7-APPswe-Eu substrate at BACE-1.
Measurement: Ki = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 471.52, ALogP 2.46, HBA 8, HBD 3, PSA 133.19, QED 0.53
Compound (SMILES): COc1ccc(C#N)cc1-n1cc(-c2cn(C)c(=O)cc2-c2ccc(Cl)cc2)cn1
InChIKey: GWBVHJMEEZKTIF-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 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 416.87, ALogP 4.44, HBA 6, HBD 0, PSA 72.84, QED 0.49
Compound (SMILES): COc1cc(Cl)cc2c1nc(N)n1nc(C3CCCN(c4cnn(C)c4)C3)nc21
InChIKey: JQGMJVAGMKWLPX-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Measurement of A2a Binding Affinity Using Radioligand Binding: 148 μL (5 μg/mL) membranes (Perkin Elmer, Cat. No. RBHA2aM400UA) and 2 μL compounds of the invention to be tested (test compound) were transferred to individual wells of a 96-well polypropylene assay plate and incubated for 15 to 30 minutes at room temperature. [3H] SCH58261 ((7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine)) was diluted in assay buffer (50 mM Tris pH 7.4, 10 mM MgCl2, 0.005% Tween20) to a concentration of 4 nM and 50 μL transferred to each well of the assay plate. To define total and non-specific binding, wells containing 1% DMSO and 1 μM ZM241385 (Tocris Bioscience, Cat. No. 1036) respectively, were also included. The assay plate was incubated at room temperature for 60 minutes with agitation. Using a FilterMate Harvester® (Perkin Elmer), the contents of the assay plate were filtered through a UniFilter-96® PEI coated plate (Perkin Elmer Cat. No. 6005274 or 6005277). Filtering was achieved by aspirating the contents of the assay plate for 5 seconds, then washing and aspirating the contents three times with ice-cooled wash buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl) and allowing the vacuum manifold to dry the plate for 30 seconds. The filter plate was incubated for at least 1 hour at 55° C., and allowed to dry. The bottom of the filter plate was sealed with backing tape. 40 μL Ultima Gold (Perkin Elmer, Cat. No. 6013329) was added to each well of the filter plate and the top of the plate was sealed with TopSeal-A PLUS® clear plate seal (Perkin Elmer, Cat. No. 6050185). The plate was incubated for at least 20 minutes, and then the amount of radioactivity remaining in each well was determined using a TopCount® (Perkin Elmer) scintillation counter. After normalization to total and non-specific binding, the percent effect at each compound concentration was calculated. The plot of percent effect versus the log of compound concentration was analyzed electronically using a 4-parameter logistic fit based on the Levenberg-Marquardt algorithm to generate IC50 values.
Measurement: IC50 = 17.7 nM (pChEMBL 7.75)
Descriptors: MW 412.89, ALogP 2.64, HBA 9, HBD 1, PSA 99.39, QED 0.55
Compound (SMILES): Cc1nc(C(C)NC(=O)CN2Cc3ccc(-c4nc(NC5CCOCC5)ncc4Cl)cc3C2=O)cs1
InChIKey: SVYKMFBGUMCLHS-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 In Vitro Inhibition Assay: Activity of ERK2 enzyme (Life Technologies) was determined using a time-resolved fluorescence format measuring the phosphorylation of a truncated version of Activating transcription factor 2 labelled with green fluorescent protein (ATF2-GFP) (Life Technologies). Assay reactions containing 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 mM ATF2-GFP, 20 μM ATP and 0.25 nM ERK2 were set up in the presence of compound and allowed to proceed for 30 min at room temperature. Reactions were then stopped using TR-FRET dilution buffer (Life Technologies), 25 mM EDTA and 2 nM Tb-Anti-pATF2 (Thr71) (Life Technologies). After a further incubation period of at least 30 minutes, fluorescence was read on a Pherastar reader (Lanthascreen optic module; excitation 340 nm, emission 520 nm (channel A), 495 nm (channel B)). The ratio between A and B counts was used to calculate signal. IC50 values were calculated using a sigmoidal dose response equation (Prism GraphPad software, La Jolla, Calif., USA).
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 527.05, ALogP 3.99, HBA 8, HBD 2, PSA 109.34, QED 0.48
Compound (SMILES): O=C(CN1CCN(c2cccc3c2cnn3-c2ccccc2F)C1=O)N1CC(F)C(F)C1
InChIKey: BQJMKCFCENTDAX-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 = 1060 nM (pChEMBL 5.96999979019165)
Descriptors: MW 443.43, ALogP 2.93, HBA 4, HBD 0, PSA 61.68, QED 0.62
Compound (SMILES): COc1cc(-c2cn(C)c(=O)c3cnccc23)cc(OC)c1CN1CC(S(=O)(=O)NCCCCCCNc2ccc3c(c2)C(=O)N(C2CCC(=O)NC2=O)C3=O)C1
InChIKey: MXASZUHWROATGE-UHFFFAOYSA-N
Target: Bromodomain-containing protein 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H8M2
Assay: Bromodomain TR-FRET Competition Binding Assay: A mixture of biotinylated-ligand and SureLight Allophycocyanin-Streptavidin (APC-SA, PerkinElmer AD0201) in 50 mM HEPES (pH 7.4), 50 mM NaCl, 1 mM TCEP (pH 7), 0.01% (v/v) Tween-20, 0.01% (w/v) bovine serum albumin was added to a white 384-well PerkinElmer Proxiplate Plus plate. DMSO or 3-fold serially diluted compounds were then added to the Proxiplate followed by addition of Flag-BRD9. After a 10-minute incubation at room temperature, Eu-W1024 anti-FLAG (PerkinElmer, AD0273) was added. The final reaction miture that contained 3.75 nM biotinylated ligand, 3 nM Flag-BRD9, 7.5 nM SureLight Allophycocyanin-Streptavidin, and 0.2 nM Eu-W1024 anti-FLAG was incubated at room temperature for 90 minutes.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 799.91, ALogP 2.8, HBA 13, HBD 3, PSA 198.34, QED 0.11
Compound (SMILES): O=C(NCc1cccc(Cl)c1Cl)[C@]1(F)CC[C@@H](O)c2ncccc21
InChIKey: RFFYIHZMHKPFCL-DYVFJYSZSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Pharmacological Assay: 1. Wash each well with 80 microL of assay buffer (20 mM HEPES, 1×HBSS, pH 7.4 adjusted with NaOH) three times and leave 20 microL using plate washer, ELx-405 Select CW (BIO-TEK).2. Add 20 microL of assay buffer containing 2.5 mM probenecid, 0.5 microM Fluo-4-AM (Molecular Probes) and 0.1% Pluronic F-127 to each well.3. Incubate the plate at 37° C. in 5% CO2 for 1 h.4. Wash each well with 80 microL of assay buffer (see below) three times and leave 20 microL using plate washer, ELx-405 Select CW (BIO-TEK).5. Test compounds were prepared at 100× the test concentration in DMSO by serial dilution with Biomek-FX liquid handling instrument. 33× diluted compound solutions in assay buffer were prepared in intermediate compound plate with Biomek-NX liquid handling instrument. A further 3× dilution occurred in below steps 6 and 7.6. Add 20 microL of 33× diluted compound solutions into each well and leave the plate for 10 min under the dark at room temperature.7. Measure activity by FDSS as follows:Set the assay plate on the stacker of FDSS.Start the detection of fluorescence intensity at 540 nm by 480 nm exicitation.After 30 seconds, add 20 microL of assay buffer containing 240 microM BzATP (final concentration 80 microM).
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 369.22, ALogP 3.7, HBA 3, HBD 2, PSA 62.22, QED 0.87
Compound (SMILES): COC(=O)c1cc(Cl)c(N)cc1S(=O)(=O)N[C@H](c1n[nH]c(=O)o1)[C@H](C)c1c(F)ccc(C)c1C
InChIKey: DCLOSQAXJCJACM-ZMZPIMSZSA-N
Target: Ribonucleotide reductase (PROTEIN COMPLEX), Homo sapiens
UniProt: P23921
Assay: Human RNR Inhibition Effect: The inhibitory activity against the ribonucleotide reduction reaction (hereinafter referred to as RNR reaction) of the test compound was determined by measuring the formation of deoxycytidine diphosphate (hereinafter referred to as dCDP) from cytidine diphosphate (hereinafter referred to as CDP) by the following method.Human M1 subunit and human M2 subunit (mutant lacking amino terminal 59 amino acids), which are fused a histidine tag at the amino terminus, are overexpressed in Escherichia coli and are solubilized after collection, and histidine tagged human M1 and M2 proteins were purified on a nickel chelate column.For measuring the inhibitory activity of the test compound against the RNR reaction, the method described in the document [CANCER RESEARCH 64, 1-6, 2004] was referred to.
Measurement: IC50 = 660 nM (pChEMBL 6.179999828338623)
Descriptors: MW 512.95, ALogP 2.96, HBA 8, HBD 3, PSA 157.38, QED 0.32
Compound (SMILES): CN(c1ccc(S(=O)(=O)Nc2nccs2)cc1Cl)[C@H]1CCN(Cc2cccc(C(F)F)c2)C1
InChIKey: ZTRURHABBFJPPY-KRWDZBQOSA-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 = 221 nM (pChEMBL 6.659999847412109)
Descriptors: MW 513.04, ALogP 5.25, HBA 6, HBD 1, PSA 65.54, QED 0.45
Compound (SMILES): Cn1cnc(S(=O)(=O)N2CC(Nc3cccc(OC(F)(F)F)c3)C(C3CCCN(C4CC4)C3)C2)c1
InChIKey: SCOWVEIVGCMGLP-UHFFFAOYSA-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 < 100 nM (pChEMBL 7)
Descriptors: MW 513.59, ALogP 3.29, HBA 7, HBD 1, PSA 79.7, QED 0.61
Compound (SMILES): COCCOc1cc2nccc(Oc3ccc(NC(=O)C4(C(=O)Nc5ccc(F)cc5)CC4)cc3F)c2c2c1OCCO2
InChIKey: QLMCQLQRMIDQIJ-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Assay of Small Molecular Compounds for Inhibiting the Activity of c-MET Kinase: The assay is based on the LANCE TR-FRET technology of Perkin Elmer Inc., and the assay method is as follows:1. Dilution of compounds: a total of 11 concentrations were obtained using a 3-fold gradient dilution from the highest concentration of 2500 nM (the maximum final concentration of the drug used in this assay was 2500 nM, and the minimum final concentration was 0.042 nM).2. 2.5 μL of the gradient-diluted compounds were taken with a transfer pipette to a 384-well plate.3. Addition of enzyme: 5 μL of 2× c-MET kinase solution (concentration was 2 nM) was taken with a transfer pipette to the corresponding reaction well of the 384-well plate, mixed well and pre-reacted at room temperature for 5 minutes.4. 2.5 μL 4× Ultra ULight-JAK-1 (Tyr1023) Peptide (concentration was 400 nM)/ATP (concentration was 40 μM) mixture 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 mixture and 7.5 μL 1× Kinase Assay Buffer were added to the wells of the 384-well plate.6. Positive control: 2.5 μL/well 4× substrate/ATP mixture, 2.5 μL/well 1× Kinase Assay Buffer containing 16% DMSO, and 5 L/well 2× c-MET kinase solution were added to the 384-well plate. The final concentration of DMSO in the reaction system was 4%.7. The mixture was mixed well and then centrifuged and reacted at room temperature in dark for 60 min.8. 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.9. Development of the reaction: 5 μL of 4× detection solution 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.10. The 384-well plate was placed into the Envision plate reader and the signal was detected using the appropriate program.11. Analysis and processing of the raw data:12. The drug concentrations and the corresponding inhibition rates were input into GraphPad Prism5 for calculation, and the inhibition rate of the compounds were calculated as follows: inhibition rate (%)=(reading of positive well−reading of experimental well)/(reading of positive control well−reading of negative control well)×100%. Processing with GraphPad Prism5 software yielded the corresponding IC50 values (the concentration of the compound at which 50% of the highest inhibition of the enzyme is achieved).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 591.57, ALogP 5.46, HBA 8, HBD 2, PSA 117.24, QED 0.18
Compound (SMILES): c1ccc(-c2cnc(-c3cc(-c4ccc(N5CCOCC5)nc4)ccn3)[nH]2)cc1
InChIKey: NQJBBDMOEGITQG-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: PI3K-γ Scintillation Proximity Assay: 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). IC50 determination was performed by fitting the curve of percent of the solvent control activity versus the log of the inhibitor concentration using the GraphPad Prism 6.0 software.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 383.45, ALogP 4.04, HBA 5, HBD 1, PSA 66.93, QED 0.58
Compound (SMILES): CN(CCC1CCOCC1)c1nc2c(c(NC3(C)CC3)n1)[S@+]([O-])CC2
InChIKey: OXEBZZKMJHCSIO-RUZDIDTESA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: PDE4 Assay: The human PDE41 Dcatalytic domain (UniProt no. Q08499 [S380-L740]) was incubated with a mixture of non-labelled cAMP (cyclic adenosine monophosphate) and fluorescein amidite (FAM) conjugated cAMP and titrated test or reference compound. Following brief incubation the enzymatic reaction was stopped by addition of binding buffer containing nanoparticles with immobilized trivalent metal ions capable of binding 1) AMP phospho groups and 2) terbium (Tb) donor fluorophores. Subsequent excitation of the Tb donor triggers time-resolved FRET to adjacent FAM acceptor molecules resulting in light emission. In the presence of a PDE4 inhibitor, AMP generation was reduced resulting in a lower fluorescence signal. The cAMP phosphodiester is not bound by the detection system.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 364.52, ALogP 2.36, HBA 6, HBD 1, PSA 73.34, QED 0.78
Compound (SMILES): CCS(=O)(=O)c1cccc(-c2c(C(C)C)n(C/C(F)=C/CN)c3ccc(C)nc23)c1
InChIKey: JMDGXURWTPBBKZ-WQRHYEAKSA-N
Target: Lysyl oxidase homolog 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y4K0
Assay: Inhibition Assay: LOX and LOXL2: Lysyl oxidase (LOX) is an extracellular copper dependent enzyme which oxidizes peptidyl lysine and hydroxylysine residues in collagen and lysine residues in elastin to produce peptidyl alpha-aminoadipic-delta-semialdehydes. This catalytic reaction can be irreversibly inhibited by β-aminopropionitrile (BAPN) that binds to the active site of LOX (Tang S. S., Trackman P. C. and Kagan H. M., Reaction of aortic lysyl oxidase with beta-aminoproprionitrile. J Biol Chem 1983; 258: 4331-4338). There are five LOX family members; these are LOX, LOXL1, LOXL2, LOXL3 and LOXL4. LOX and LOXL family members can be acquired as recombinant active proteins from commercial sources, or extracted from animal tissues like bovine aorta, tendons, pig skin; or prepared from cell cultures. The inhibitory effects of the compounds of the present invention were tested against the given LOX-LOXL preparation using a high-throughput coupled colorimetric method (Holt A. and Palcic M., A peroxidase-coupled continuous absorbance plate-reader assay for flavin monoamine oxidases, copper-containing amine oxidases and related enzymes. Nat. Protoc. 2006; 1: 2498-2505). The assay was developed using either 384 or 96 well format. Briefly, in a standard 384 well plate assay 25 μL of a dilution of any of the isoenzymes and orthologues in 1.2 M urea, 50 mM sodium borate buffer (pH 8.2) were added into each well in the presence of 1 μM mofegiline and 0.5 mM pargyline (to inhibit SSAO and MAO-B and MAO-A, respectively). Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 11 data points, typically in the micromolar or nanomolar range after incubation with the enzyme for 30 min at 37° C. Twenty five L of a reaction mixture containing twice the KM concentration of putrescine (Sigma Aldrich, e.g. 20 mM for LOX, or 10 mM for LOXL2 and LOXL3), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 1.2 M urea, 50 mM sodium borate buffer (pH 8.2) were then added to the corresponding wells. The above volumes were doubled in the case of 96 wells plate.
Measurement: IC50 < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 429.56, ALogP 4.74, HBA 5, HBD 1, PSA 77.98, QED 0.59
Compound (SMILES): CNCC[C@H](Oc1ccc(CN2CCN(C)c3nc(N)ccc3C2=O)cc1)c1cccs1
InChIKey: XMJZVIRIHVLEIF-FQEVSTJZSA-N
Target: Voltage-dependent calcium channel subunit alpha-2/delta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P54289
Assay: Binding Assay : Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4.NSB (non specific binding) was measured by adding 10 μM pregabalin. The binding of the test compound was measured in five different concentrations. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4.Filter plates were dried at 60° C. for 1 hour and 30 μl of scintillation cocktail were added to each well before radioactivity reading.Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 451.6, ALogP 3.55, HBA 7, HBD 2, PSA 83.72, QED 0.55
Compound (SMILES): CC(C)Oc1nc2c(cc1Nc1nccc(-c3cc(C#N)c4c(c3)[C@@](C)(CO)CN4)n1)CN(C1CC1)CC2
InChIKey: RNKVENHAUSXFKH-GDLZYMKVSA-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 = 34.7 nM (pChEMBL 7.460000038146973)
Descriptors: MW 511.63, ALogP 4.14, HBA 9, HBD 3, PSA 119.22, QED 0.43
Compound (SMILES): COc1ccc(C(=O)Nc2ccc(-c3ccccc3F)nc2)cc1NC(=O)C1(N2CCOCC2)CC1
InChIKey: OYXSUAHLPHEGQU-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 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 490.54, ALogP 3.95, HBA 6, HBD 2, PSA 92.79, QED 0.52
Compound (SMILES): CC1Cc2cc(-c3cccnc3)ccc2C(c2c(F)cc(/C=C/C(=O)O)cc2F)N1CC(C)(C)F
InChIKey: PZIJVZDKKQUGLE-RMKNXTFCSA-N
Target: G-protein coupled estrogen receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99527
Assay: Biological Assay: The compounds of the present invention have an inhibition effect on the binding of E (estrogen) to ER (estrogen receptor), thereby blocking the binding of a complex of E and ER to ERE (estrogen responsive element), and subsequently blocking the expression of downstream luciferase protein.
Measurement: IC50 = 2.28 nM (pChEMBL 8.640000343322754)
Descriptors: MW 480.53, ALogP 6.21, HBA 3, HBD 1, PSA 53.43, QED 0.43
Compound (SMILES): C=CC(=O)Nc1ccc(Oc2ccc(F)cc2F)c(-c2cn(C)c(=O)c3ccccc23)c1
InChIKey: NESJFIVVYHTKBZ-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Binding Assay: The bromodomain binding assays were performed by Reaction Biology Corp., Malvern, Pa., USA (www.reactionbiology.com). The BET binding assays were conducted in 384 well microplates in assay buffer (50 mM HEPES-HCl, pH 7.5, 100 mM NaCl, 1 mg/ml BSA, 0.05% CHAPS, and 0.5% DMSO) with compounds added as DMSO stocks at a single concentration or with 10-point dose response titrations. BET protein or assay buffer were delivered to the appropriate wells of the microplate. Test compound was then delivered by acoustic technology via a Labcyte Echo550 liquid handler. The microplate was centrifuged for 5 min and pre-incubated for 30 min at RT with gentle shaking. The ligand (histone H4 peptide (1-21) K5/8/12/16Ac-biotin) was delivered and the microplate was again centrifuged for 5 min and allowed to incubate for 30 min at RT with gentle shaking. Donor beads were then added in the absence of light and the microplate was centrifuged and gently shaken. After 5 min, acceptor beads were added in the absence of light and the microplate was centrifuged and gently shakenin the dark for 60 min. The microplate was read using a Perkin Elmer EnSpire Alpha plate reader (λEx/λEm=680/520-620 nm). Percent inhibition was calculated relative to positive and negative controls on a per plate basis.
Measurement: IC50 = 319 nM (pChEMBL 6.5)
Descriptors: MW 432.43, ALogP 5.4, HBA 4, HBD 1, PSA 60.33, QED 0.43