lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(NS(=O)(=O)c1ccc(NCC2(F)CCOCC2)c([N+](=O)[O-])c1)c1ccc(C2=CCC(N3CCC[C@@H]3c3ccccc3C3CC3)CC2)cc1Oc1cnc2[nH]ccc2c1
InChIKey: YIOIFXQIHFPTCA-RUPAOVHOSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Bcl-2/Bcl-X Fluorescence Polarization Assay: Compounds disclosed herein were tested for blocking of Bcl-2/Bcl-xl protein with its ligand in an assay based on fluorescence polarization methodology. Recombinant human 2.7 nM Bcl-2/1.3 nM Bcl-xl protein was pre-incubated with a serial dilution of compounds disclosed herein (maximum concentration is 1 μM for Bcl-2 assay, and 10 μM or 1 μM for Bcl-xl assay, 3-fold serially diluted, 10 points) at room temperature for 0.5 hour in an assay buffer containing 20 ml potassium phosphate buffer, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.05% Tween-20, 0.01% BSA. Then the FITC labeled Bak peptide Ac-GQVGRQLAIIGDK (FITC)INR-amide (1 nM for Bcl-2, 0.82 nM for Bcl-xl) was added to plate and further incubated at room temperature for 0.5 h. The FP signals (485 nm-520 nm-520 nm) were read on BMG PHERAstar FS or BMG PHERAstar FSX instalment. The inhibition percentage of Bcl-2/Bcl-xl interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the FP signals. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Measurement: IC50 = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 834.97, ALogP 8.96, HBA 10, HBD 3, PSA 168.79, QED 0.08
Compound (SMILES): CCS(=O)(=O)Nc1cc(-c2nc3ccc(F)cc3n2C2CC2)cnn1
InChIKey: ALCSVNKNTSIYLR-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Metalloenzyme Activity: V79-4 cells expressing recombinant andrenodoxin and andrenodoxin reductase with either recombinant human CYP11B2 or CYP11B1 were prepared according to methods previously described (LaSala et al 2009 Anal Bioch 394:56-61). An enzyme enriched microsomal fraction was prepared from cellular lysates and subsequently used as the enzyme source for determining inhibitor IC50s. The substrate Km values were experimentally determined for 11-deoxycorticosterone (CYP11B2 substrate) and 11-deoxycortisol (CYP11B1 substrate). Enzyme assays for inhibitor screening employed CYP11B2 and CYP11B1 enzyme enriched microsomes and were run at the Km of the respective substrates. Products of the enzyme reactions, aldosterone for CYP11B2 or cortisol for CYP11B1, were measured by LC-MS. Assays were run under conditions of less than 20% substrate turnover. Inhibitor IC50s were generated by determining the product formation in the absence or presence of inhibitor at various concentrations. In the absence of the test compound, the product formed (Pt) in each data set was defined as 100% activity. In the absence of enzyme, the product formed (Pb) in each data set was defined as 0% activity. The percent activity in the presence of each inhibitor was calculated according to the following equation: % activity=(P−Pb)/(Pt−Pb), where P=the product formed in the presence of the inhibitor. The IC50 value was defined as the inhibitor concentration causing a 50% decrease in activity relative to the no inhibitor control reaction.
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 361.4, ALogP 2.73, HBA 6, HBD 1, PSA 89.77, QED 0.75
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CN(C(=O)CC2CCCCC2)c2ccccc2N(Cc2c(C)ccc3ccccc23)C1=O
InChIKey: JWZHGQQKGZBONI-FIPFOOKPSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay for BIR2 and BIR3: Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(c-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations. Percentage of inhibition at each concentration was determined to generate an IC50 value for each test compound.
Measurement: IC50 = 685 nM (pChEMBL 6.159999847412109)
Descriptors: MW 540.71, ALogP 5.09, HBA 4, HBD 2, PSA 81.75, QED 0.44
Compound (SMILES): Cc1cc(C#N)ccc1-c1cnn(-c2ccc(C(=O)NC3CCOCC3)cn2)c1O
InChIKey: NQNPQFMINISIIP-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Inhibition Assay: The IC50 values for the PHD2 enzyme (residues 181-417) were determined by mixing increasing amounts of inhibitor with a fixed amount of enzyme (5 nM, final concentration) and Biotin labeled peptide (Biotin-Asp-Leu-Glu-Met-Leu-Ala-Pro-Tyr-Ile-Pro-Met-Asp-Asp-Asp-Phe-Gln-Leu, 1 uM final concentration) and 2-Oxyglutarate (2 uM final concentration) in 50 mM HEPES, 50 mM KCl, 0.5 mM TCEP, 2 uM FeCl2, 0.1 mg/ml BSA, at pH 7.3. The reaction was conducted by pre-incubating the enzyme in the presence of inhibitor for 60 min at room temperature. The activity of the free enzyme was measured by adding the peptide, the 2-Oxoglutarate (see above for final concentrations), and Ascorbic Acid (1 mM final concentration). The enzymatic activity was quenched after 60 min by adding an excess of a tight binding inhibitor to the assay mixture. The amount of product released was measured by using a LC/MS system (Agilent HPLC with Applied Biosystems API3000 Mass Spectrometer).
Measurement: IC50 = 46.8 nM (pChEMBL 7.329999923706055)
Descriptors: MW 403.44, ALogP 2.73, HBA 7, HBD 2, PSA 113.06, QED 0.69
Compound (SMILES): CC(C)(C)CS(=O)(=O)Nc1ccc(-c2cc(NC(=O)C3CC3)nc3[nH]ccc23)cc1F
InChIKey: IKXCSWUZKQQQMG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: ADP-Glo Kinase Assay: A control material and a test material were prepared for each concentration, in such a way that they were diluted by means of DMSO. At the same time, ATP (250 uM) and JAK substrate (JAK1, IRS-itide 40 ng/mL) were prepared, in such a way that they were diluted by means of kinase buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.5 mg/mL BSA, 50 uM DTT).A test drug for each concentration, substrate, ATP and JAK enzyme were mixed in an eppendorf tube, and reacted in a 30° C. incubator for 40 minutes.ADP-Glo reagent contained in ADP-Glo Kinase Enzyme System (Promega, USA, V9571) was added into each eppendorf tube, and reacted in a 30° C. incubator for 40 minutes. A kinase detection reagent contained in ADP-Go Kinase Enzyme System was inserted into the eppendorf tube, then an integration time was set to 1 second by using Wallac Victor 2, then luminescence was measured, and then JAKs phosphorylation inhibition ability of the test material was analyzed. The concentration of the compound, which causes 50% of the JAK enzyme activity inhibition in comparison With the control group, was determined as IC50 (nM) of an inhibitor, wherein the results thereof are shown in a following table 1.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 444.53, ALogP 4.51, HBA 4, HBD 3, PSA 103.95, QED 0.52
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CN(C(=O)c2ccc(C(C)=O)cc2)c2ccccc2N(Cc2c(OCC(F)(F)F)ccc3ccccc23)C1=O
InChIKey: URMJPEDUVJVTIM-KMRXNPHXSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay for BIR2 and BIR3: Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(c-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations. Percentage of inhibition at each concentration was determined to generate an IC50 value for each test compound.
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 646.67, ALogP 5.27, HBA 6, HBD 2, PSA 108.05, QED 0.24
Compound (SMILES): Cc1[nH]ncc1[C@@H]1CN(c2ccnc(-c3cnc4ccc(C(F)(F)F)cn34)n2)C[C@H](C)O1
InChIKey: MYIOCDXMLIMTFT-SJCJKPOMSA-N
Target: eIF-2-alpha kinase GCN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P2K8
Assay: Enzyme Inhibition Assay: An assay buffer containing GCN2 kinase and (RS)7 was prepared. 4.7 μL of this stock solution was placed per well of a black, low volume, 384-well microtitre plate (e.g. catalogue number 3676, Corning Inc., NY). To this was added 0.65 μM of DMSO containing serial dilutions of the test compound (typical final concentrations of test compound were 0 to 8 μM). The plate was incubated for 10 minutes at 25° C. prior to the addition of 4.7 μL of ATP stock buffer to initiate the enzyme reaction. The reaction was allowed to proceed for 1 hour at 25° C., prior to the addition of 10 μL detection buffer (consisting of appropriate concentrations of ADP2 antibody and ADP Alexa633 tracer in 1× stop and detect buffer as supplied by BellBrook Labs). The reaction was left to incubate for 1 hour at 25° C., prior to measuring the fluorescence polarisation signal (mP) in each well using a PHERAstar FS reader (BMG Labtech, Germany).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 443.43, ALogP 3.81, HBA 7, HBD 1, PSA 84.23, QED 0.52
Compound (SMILES): CC(C)Oc1ccc(C(=N)c2nc(N3CC[C@]4(CCN(CC(=O)N5CC=C(c6ccc(C(=N)/N=C\NCCO)cc6)CC5)C4)C3=O)ccc2N)cn1
InChIKey: LFQIPIPVSRTTDE-LHEWISCISA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Kinase Assay: Activated ERK1 and ERK2 activity was determined in a Mobility Shift Assay (MSA) format as follows: Compound and kinase solution were prepared with assay buffer (20 mM HEPES, 0.01% Triton X-100, 2 mM DTT, pH7.5) and mixed and incubated in for 30 mins at rt. ERK1 & ERK2 were then activated by the addition of F1-Substrate, ATP and metal solution and incubated for 1 h at rt. After 1 h, the reaction was terminated by the addition of 70 mL of Termination Buffer (QuickScout Screening Assist MSA; Carna Biosciences) to the well. The reaction mixture was applied to LabChip system (PerkinElmer), and the product and substrate peptide peaks were separated, analyzed and quantitated. The kinase reaction is evaluated by the product ratio calculated from peak heights of product (P) and substrate(S) peptides (P/(P+S)).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 706.85, ALogP 2.94, HBA 10, HBD 5, PSA 197.21, QED 0.11
Compound (SMILES): COc1cccc(F)c1C(=O)NC1CCN(C(=O)c2cc(C(C)C)[nH]n2)C1
InChIKey: ZMJWTXJSBSTDJI-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5A (SINGLE PROTEIN), Homo sapiens
UniProt: P29375
Assay: KDM5 Enzyme Assay: Full length KDM5A enzyme was expressed and purified inhouse. Biotin-H3K4me3 peptide was purchased from New England Biolabs. HTRF reagents (containing Eu-labeled H3K4me1-2 antibody, and streptavidin-XL665) were purchased from Cis-Bio International. Plates were read on an Envision multi-label plate reader (Perkin Elmer).The HTRF assay mixture contained 2 nM full length KDM5A enzyme, 100 nM H3K4Me3 peptide substrate, 1 uM 2-OG, 100 uM Fe2+, 500 uM ascorbate. 50 mM HEPES pH 7.0 buffer, 0.01% Triton-X 100, 2 mM DTT, 0.25% DMSO at a final volume of 10 uL.The enzyme reaction was carried out at room temperature in black Proxiplate 384-Plus plate (Corning. Costar) within 30 minutes, in the presence of varying concentration of a test compound. At the end of enzyme reaction, 5 uL of 1 mM EDTA were added to quench the reaction and then the detection reagents (5 uL) were added to give final concentrations of 0.5 nM Eu-labeled H3K4me1-2 antibody, and 50 nM streptavidin-XL665. The plates were incubated at room temperature for 60 minutes and then read in the Envision plate reader. The readouts were transformed into % inhibition, and IC50 value of a test compounds was generated by using four parameters curve fitting (Model 205 in XLFIT5, iDBS).
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 374.42, ALogP 2.33, HBA 4, HBD 2, PSA 87.32, QED 0.84
Compound (SMILES): Cc1nccc(Nc2cc(-c3cnc(C)c(OC4CCCC4)c3)ccn2)n1
InChIKey: MGRHSNQEMSKJFW-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase catalytic subunit type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NEB9
Assay: VPS34 Enzyme Assays: 100 nL compounds in DMSO are added to wells of a 384 well microtitre plate (Greiner 780076). At room temperature: 5 ul VPS34 reaction buffer (Invitrogen Assay Buffer Q (diluted 1 in 5 with nanopure water) plus 2 mM DTT and 2 mM MnCl2) containing ATP (20 uM, Promega) and 200 uM PI-PS substrate (Invitrogen PV5122) is added followed immediately by 5 ul VPS34 reaction buffer (as above) containing VPS34 (5 nM, Millennium Protein Sciences Group) and the mixture is incubated with shaking at room temperature for 1 hour. Then 5 ul VPS34 stop-detect mix (as per Invitrogen Adapta Assay kit (PV5009) instructions (contains kinase quench buffer, TR-FRET buffer, Adapta Eu anti-ADP antibody and Alexa Fluor 647 ADP tracer)) is added to quench the reaction. The plates are then incubated for 30 minutes at room temperature with shaking and then read on a BMG PheraStar Plus reader.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 361.45, ALogP 4.62, HBA 6, HBD 1, PSA 72.82, QED 0.71
Compound (SMILES): CC(=O)N[C@@H](C)CO[C@H]1CC[C@H](Oc2ccc3nc(C(C)(C)O)sc3c2Cl)CC1
InChIKey: AADYGOAJMVENRH-QEJZJMRPSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Inhibitory Activity on Human ACC1 and the ACC2: Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H]−+Intensity of [AcCoA-H]−) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
Measurement: IC50 = 336 nM (pChEMBL 6.46999979019165)
Descriptors: MW 440.99, ALogP 4.41, HBA 6, HBD 2, PSA 80.68, QED 0.67
Compound (SMILES): C[C@]1(O)C[C@@H](c2nc(-c3ccc(Oc4cc(F)cc(OCc5ccccc5)c4)cc3)c3c(N)nccn32)C1
InChIKey: BGJYGCPOOTWEME-IXKSNFPSSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Inhibition Assay: Method A: Fluorescence polarization-based kinase assays were performed in 384 well-plate format using histidine tagged recombinant human full-length Bruton Agammaglobulinemia Tyrosine Kinase (Btk) and a modified protocol of the KinEASE FP Fluorescein Green Assay supplied from Millipore®. Kinase reaction were performed at room temperature for 60 minutes in presence of 250 μM substrate, 10 μM ATP and variable test article concentrations. The reaction was stopped with EDTA/kinease detection reagents. Phosphorylation of the substrate peptide was detected by fluorescence polarization measured with a Tecan 500 instrument.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 510.57, ALogP 6.12, HBA 7, HBD 2, PSA 94.9, QED 0.27
Compound (SMILES): Cc1nc(C#Cc2cccc(C(=O)Nc3ccc(C(F)(F)F)cc3)c2)cs1
InChIKey: FLGBIOCDQABTQW-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: In Vitro Activity Test for mGluR5: Experimental material: HEK293/mGluR5 cell line, Fluo-8 calcium ion fluorescent dye, positive control MPEP, CTEPExperimental instrument: FLIPR Tetra real-time fluorescence imaging analysis systemExperimental method: HDB Fluo-8 calcium fluorescence detection methodExperimental principle: HDB Fluo-8 calcium ion fluorescence detection method is a fast, simple and reliable fluorescence detection method of intracellular calcium concentration changes. The Fluo 8-AM fluorescent dye is an acetyl methyl ester derivative of Fluo 8 which can easily penetrate the cell membrane into the cell by culture. The fluorescent dye into the cell will be hydrolyzed by intracellular esterase, the resulting Fluo 8 cannot easily pass through the lipid bimolecular membrane as a polar molecule, and will retain in the cell, and combine with calcium (Ca2+) to produce fluoresces.Cells expressing GPCR receptor protein (mGluR5) were first calibrated with a calcium ion sensitive fluorescent probe and then stimulated with the compound. After stimulation, the activation of the receptor lead to the calcium ion mobilization, and the fluorescent probe captures the calcium ion to induce the fluorescence signal. The signal can be read by a fluorescent plate reader. The fluorescent plate reader contains a sampling head for compound addition, thus enabling the change of the fluorescence value of the compound be read in real time. If the selected compound can activate mGluR5, the calcium flow reaction can be greatly increased; conversely, if the selected compound is able to antagonize mGluR5, the calcium flow reaction can be greatly reduced.
Measurement: IC50 = 29200 nM (pChEMBL 4.539999961853027)
Descriptors: MW 386.4, ALogP 5.12, HBA 3, HBD 1, PSA 41.99, QED 0.62
Compound (SMILES): O=C(Oc1ccccc1)N1CCC[C@@H]1c1nc2c(=O)[nH]c(-c3ccccc3F)cc2s1
InChIKey: BCSFKCDCDLTLQE-GOSISDBHSA-N
Target: Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q8N5Z0
Assay: KAT-II inhibitory assay: The inhibitory action of the test compound on human recombinant KAT-II was determined by the following method.To a reaction mixture (45 μL) containing 3.0 μmol/L kynurenine, 10 μmol/L pyridoxal phosphate, 2.0 ng/μL human recombinant KAT-II, and 150 mmol/L tris(hydroxymethyl)aminomethane-acetate buffer (pH 8.0) was added a 10% dimethyl sulfoxide solution (5 μL) of each test compound prepared, and the mixture was reacted at 37° C. for 1 hr. After the reaction, 50% trichloroacetic acid (5 μL) was added to terminate the reaction.The resultant kynurenic acid was quantified as follows by high performance liquid chromatography. An enzyme reaction mixture was separated by an octadecylsilane reversed-phase column (SC-50DS, Eicom Corporation; mobile phase: 250 mmol/L zinc acetate, 50 mmol/L sodium acetate, and 5.0% acetonitrile (pH 6.2)) incubated at 30° C., and kynurenic acid was quantified using a fluorescence detector (RF-20Axs, Shimadzu Corporation) at excitation wavelength 354 nm, detection wavelength 460 nm. The analytical curve was drawn every time by an external standard method. Each test compound was tested by dual measurement at each concentration. The kynurenic acid level in the presence of a test compound at each concentration was converted into % relative to kynurenic acid resulting from a reaction with an enzyme alone as 100%, and the obtained values were fitted to S-curve to determine IC50.
Measurement: IC50 = 69 nM (pChEMBL 7.159999847412109)
Descriptors: MW 435.48, ALogP 5.13, HBA 5, HBD 1, PSA 75.29, QED 0.48
Compound (SMILES): CN(C)Cc1c(C(F)(F)F)cc2ncn(CCOc3ccc(Cl)cc3-c3ccnc4c(C(=O)O)csc34)c(=O)c2c1C#N
InChIKey: SEQYTUHIRYAMHI-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 73 nM (pChEMBL 7.139999866485596)
Descriptors: MW 628.03, ALogP 6.06, HBA 9, HBD 1, PSA 121.34, QED 0.22
Compound (SMILES): NS(=O)(=O)c1c(S(=O)(=O)C2CNC2)ccc(-c2cccc3nccn23)c1-c1nnn[nH]1
InChIKey: MLYDAXVVMRBRMI-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Enzyme Activity: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1; for a review, see: Meine, M.-R.; Llarrull, L. I.; Vila, A. J. Antibiotics, 2014, 3, 285-316) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 μM to 0.00063 μM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 0.591 nM (pChEMBL 9.229999542236328)
Descriptors: MW 460.5, ALogP -0.43, HBA 10, HBD 3, PSA 178.09, QED 0.36
Compound (SMILES): CC(C)(C(=O)O)c1cccc(S(=O)(=O)CCCCOc2ccc(-c3ccn[nH]3)nc2)c1
InChIKey: NMEDQBVCAPEMRM-UHFFFAOYSA-N
Target: Cytochrome P450 4F2 (SINGLE PROTEIN), Homo sapiens
UniProt: P78329
Assay: Inhibition Test for Each Compound of the Present Invention Against 20-HETE Producing Enzymes (CYP4F2 and CYP4A11): In the CYP4F2 inhibition test, the reaction solution containing each compound [final concentration of 50 mM, KPO4 (pH 7.4), 2.5 μM luciferine derivative, and 1 mM NADPH] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4F2 had been expressed. In the CYP4A11 inhibition test, the reaction solution containing each compound [final concentration of 100 mM, Tris-HCl (pH 7.5), 60 μM luciferine derivative, 1.3 mM NADP+, 3.3 mM Glucose 6-Phosphate, 3.3 mM MgCl2, and 0.4 U/mL Glucose 6-Phosphate dehydrogenase] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4A11 had been expressed. Following this, the membrane fraction was left to stand at room temperature for 60 minutes to perform an enzymatic reaction. After the reaction, a luciferine detection reagent was added, and the luminescence value was measured using a plate reader. By using that value, the percent inhibition of 20-HETE producing enzyme (%) was calculated according to the equation described below, and the 50% inhibitory concentration (IC50 value) for each compound was calculated.
Measurement: IC50 = 2300 nM (pChEMBL 5.639999866485596)
Descriptors: MW 443.53, ALogP 3.47, HBA 6, HBD 2, PSA 122.24, QED 0.46
Compound (SMILES): Cc1nn(C2CC2)c(C)c1-c1cnc(NCc2c(F)ccc3c2CCO3)n2cnnc12
InChIKey: AQMDZAWSEXSOPJ-UHFFFAOYSA-N
Target: Polycomb protein EED (SINGLE PROTEIN), Homo sapiens
UniProt: O75530
Assay: EED-H3K27Me3 Peptide Competition Binding Assay by AlphaScreen: To assess the compounds potency in the EED-H3K27Me3 competition binding assay, compounds were serially diluted 3-fold in DMSO to obtain a total of twelve concentrations. Then compounds at each concentration (75 nL of each) were transferred by Mosquito into a 384-well Perkin Elmer ProxiPlate 384 plus plates. 8 uL of solutions containing 30 nM EED (1-441)-His protein and 15 nM biotin-H3K27Me3 (19-33) peptide in the buffer (25 mM HEPES, pH 8, 0.02% Tween-20, 0.5% BSA) were added to the wells and then incubated with compound for 20 min. AlphaScreen detection beads mix was prepared immediately before use by mixing nickel chelate acceptor beads and streptavidin donor beads in a 1:1 ratio (Perkin Elmer, Product No. 6760619C/M/R) into the buffer described above. Then 4 μL of detection beads mix was added to the plate and incubate in the dark at the rt for 1 h. The final concentration of donor and acceptor beads was 10 μg/mL for each. Plates were read on EnVision (PerkinElmer) using the AlphaScreen setting adapted for optimal signal detection with a 615 nm filter, after sample excitation at 680 nm. The emission signal at 615 nm was used to quantify compounds inhibition. AlphaScreen signals were normalized based on the reading coming from the positive (maximum signal control) and negative controls (minimum signal control) to give percentage of activities left. The data were then fit to a dose response equation using the program Helios (Novartis) to get the IC50 values. Helios is a Novartis in-house assay data analysis software using the methods described by Normolle, D. P., Statistics in Medicine, 12:2025-2042 (1993); Formenko, I. et al, Computer Methods and Programs in Biomedicine, 82, 31-37 (2006); Sebaugh, J. L., Pharmaceutical Statistics, 10:128-134 (2011); Kelly, C. et al., Biometrics, 46(4):1071-1085 (1990); and Kahm, M. et al., Journal of Statistical Software, 33(7): (2010) (grofit: Fitting Biological Growth Curves with R, pages 1-21, available at http://www.jstatsoft.org/).Each compound was counterscreened to determine if it interfered with the AlphaScreen beads. Compounds were diluted as described in the preceding section, and the assay was performed by adding 12 μL of 10 nM biotin-miniPEG-His6 peptide in the above buffer and incubating for 20 min at rt prior to addition of the beads to 10 μg/mL each. The plates were then incubated for 1 h at rt in dark before being read on EnVison.
Measurement: IC50 = 12.4 nM (pChEMBL 7.909999847412109)
Descriptors: MW 419.46, ALogP 3.63, HBA 8, HBD 1, PSA 82.16, QED 0.53
Compound (SMILES): COc1ccc(CCCCCCCCCCN=C=S)cc1
InChIKey: JIINHCDTBTUHBS-UHFFFAOYSA-N
Target: N-acylethanolamine-hydrolyzing acid amidase (SINGLE PROTEIN), Homo sapiens
UniProt: Q02083
Assay: Inhibition Assay: In order to have an assay method more conducive to high-throughput screening than those published for measuring the NAE hydrolyzing activity of NAAA, we developed the fluorogenic PEA analog N-(4-methyl coumarin)palmitamide (PAMCA), which is hydrolyzed to fluorescent 7-amino-4-methyl coumarin (AMC) and palmitic acid. For three point concentration inhibition assays with hNAAA the following procedure is used. Purified activated NAAA (final concentration of 0.25 μg/mL) is incubated in assay buffer (100 mM citrate-phosphate buffer, pH 4.5, 3 mM DTT, 0.1% Triton X-100, 0.05% BSA, and 150 mM NaCl) made up to a total volume of 180 μL, followed by addition of the compound dissolved in 10 μL DMSO (along with DMSO neat for the control sample) with the final concentrations for each compound of 100, 10, and 1 μM, in triplicate on a 96 well plate. These samples are allowed to incubate for 15 min at room temperature and then 10 μL of a PAMCA stock solution in DMSO (final PAMCA concentration [5 μM]) was added. After 5 minutes of agitation on a shaking plate, the reaction is allowed to proceed at 37° C. for 120 minutes, with fluorescence readings taken every 10 minutes at a wavelength of 460 nm (using an excitation wavelength of 360 nm) on a Synergy HT Plate Reader using Gen5 software from Bio-Tek. The enzyme activity is calculated by converting the relative fluorescence units to AMC formed, using a standard curve of AMC. For compounds that inhibit hNAAA in range IC50 <1 μM full inhibition curves using eight different concentrations of inhibitor (8 point assay) are generated. The assay procedure used is the same as the three point assay. For ostensibly covalent compounds (as observed by a significant decrease in the slope of a plot of fluorescence vs. time in three point screen) samples are allowed to incubate for 2 hours at 37° C., instead of 15 minutes, before addition of 10 μL of a PAMCA stock solution in DMSO for a final PAMCA concentration of 5 μM. After 5 minutes of agitation on a shaking plate, the reaction is allowed to proceed at 37° C. for 120 minutes Inhibition constants are calculated using pro Fit software (Quantum Soft, Uetikon am See, Switzerland) and a Levenberg-Marquardt algorithm.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 305.49, ALogP 5.46, HBA 3, HBD 0, PSA 21.59, QED 0.29
Compound (SMILES): Cc1nc(OC2CCOC(C)(C)C2)c2[nH]c(N)c(S(=O)(=O)c3ccccc3F)c2n1
InChIKey: LRYKABOCLKXRLS-UHFFFAOYSA-N
Target: Free fatty acid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O15552
Assay: Ca-Flux Functional Assay: GPR43 agonist/PAM activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by FLIPR (manufactured by Molecular Devices). GPR43 mediated increases in intracellular Ca2+ concentration were readily detected upon activation with sodium acetate. Prior to the assay (24 hours), CHO-K1 Gα16 cells stably expressing human GPR43 were-seeded in cell culture medium in black, clear-bottom 384-well plates (Corning Inc) and grown overnight at 37° C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (Molecular Devices) diluted in HBSS containing 25 mM HEPES, 2.5 mM Probenecid, 0.1% BSA for 1 hour at 37° C., 5% CO2. 10 point half log concentration response curves of sodium acetate from 10 mM were conducted prior to the testing of compounds to calculate the sodium acetate concentration that produces 20% of the maximal response (EC20). Test compounds (at 10 point half log concentration response curves from 10 μM) were added in the presence of sodium acetate to achieve a final concentration that produces approximately 20% maximal response as calculated from the previous experiment. The changes in fluorescent signal were monitored by FLIPR upon addition of the compound/EC20 sodium acetate mix. The EC50 values were determined from ten point concentration response curves. Curves were generated using the average of two wells for each data point.
Measurement: EC50 = 63 nM (pChEMBL 7.199999809265137)
Descriptors: MW 434.49, ALogP 3.16, HBA 7, HBD 2, PSA 120.19, QED 0.65
Compound (SMILES): C=CC(=O)N1C[C@@H](C)N(c2cnc3[nH]cc(C(=O)NC(C)C)c3n2)C[C@H]1C
InChIKey: HVPDGTQIFMTGNL-CHWSQXEVSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 4 μM ATP and 1 μM peptide substrate. The JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 4.9 nM (pChEMBL 8.3100004196167)
Descriptors: MW 370.46, ALogP 1.71, HBA 5, HBD 2, PSA 94.22, QED 0.8
Compound (SMILES): CCn1nc(CCCOC(=O)c2cnc(C)o2)c2c1C(=O)NCC1(CCOCC1)C2
InChIKey: MISUHUFELHHETA-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: PDE4 Assay: The human PDE4D catalytic 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.The results were calculated as the molar concentrations resulting in 50% inhibition of the substrate cleavage compared to controls samples, and are expressed as a range of IC50 (nM).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 416.48, ALogP 2.07, HBA 8, HBD 1, PSA 108.48, QED 0.57
Compound (SMILES): C[C@@H]1Cc2c([nH]c3ccccc23)[C@@H](c2cnc(NCCNCCCF)cc2F)N1CC1(F)COC1
InChIKey: CDSCAISALTWXIW-CRICUBBOSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: ERalpha Binding Assay: Compounds were dosed directly from a compound source microplate containing serially diluted compound (4 wells containing 10 mM, 0.1 mM, 1 mM and 10 nM final compound respectively) to an assay microplate using the Labcyte Echo 550. The Echo 550 is a liquid handler that uses acoustic technology to perform direct microplate-to-microplate transfers of DMSO compound solutions and the system can be programmed to transfer multiple small nL volumes of compound from the different source plate wells to give the desired serial dilution of compound in the assay which is then back-filled to normalise the DMSO concentration across the dilution range.In total 120 nL of compound plus DMSO were added to each well and compounds were tested in a 12-point concentration response format over a final compound concentration range of 10, 2.917, 1.042, 0.2083, 0.1, 0.0292, 0.0104, 0.002083, 0.001, 0.0002917, 0.0001042, and 0.00001 μM respectively. TR-FRET dose response data obtained with each compound was exported into a suitable software package (such as Origin or Genedata) to perform curve fitting analysis. Competitive ER alpha binding was expressed as an IC50 value. This was determined by calculation of the concentration of compound that was required to give a 50% reduction in tracer compound binding to ER alpha-LBD.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 487.57, ALogP 4.14, HBA 5, HBD 3, PSA 65.21, QED 0.38
Compound (SMILES): CC(=O)N[C@H]1CCN(C(=O)c2nnc(-c3cnc(-c4ccc5cc(C#N)cnn45)cc3NC(C)C)s2)C1
InChIKey: QWNFOXILQXPWQA-KRWDZBQOSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Biochemical Assay: IRAK4 enzyme (Carna Biosciences, Chuo-ku, Kobe, Japan) activity was measured by detecting phosphorylated peptide substrate formation using an antibody against the phosphorylated peptide substrate. This is a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay, based on the STK1 KinEASE Assay (Cisbio, Bedford, Mass.). The assay was designed as a simple two-step, endpoint assay (a 5 μl enzyme reaction followed by 5 μl stop and detect Solution) performed in ProxiPlate-384 Plus plates (Perkin Elmer, Waltham, Mass.). Staurosporine, a non-selective kinase inhibitor was used as a positive control. Compounds diluted in DMSO were spotted into 384 well plates using a Labcyte® Echo 550 Liquid Handling System prior to addition of IRAK4 enzyme and peptide substrate. Reaction solutions were delivered using a Multi-Flo (Bio-Tek Instruments). The enzyme and peptide solution was incubated with compound for 15 minutes at room temp before the reaction was initiated by the addition of ATP. The standard 5 μl reaction mixture contained 500 μM ATP, 2 M peptide (STK1 Peptide), 0.75 nM of IRAK4 in reaction buffer (50 mM HEPES, pH 7.0, 0.02% NaN3, 0.01% BSA, 0.1 mM Orthovanadate, 5 mM MgC2, 0.025% NP-40, 1 mM DTT). After 120 min of incubation at room temperature, 5 μl of Stop and Detect Solution (1:100 Cryptate labeled anti-phosphorylated peptide antibody solution and 125 nM Tracer in a 50 mM HEPES pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for 60 minutes at room temperature and read on Envision 2103 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340 nm/615 nm665 nm, respectively. Fluorescence intensities at 615 nm and 665 nm emission wavelengths were expressed as aratio (665 nm/615 nm). Percentage of inhibition was calculated as below:% Inhibition=100×(Ratiosample−Ratio0% Inhibition)/(Ratio100% Inhibition−Ratio0% Inhibition)The 0% inhibition value comes from control wells lacking inhibitor. The 100% inhibition value comes from control wells containing a saturating amount of known inhibitor staurosporine.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 515.6, ALogP 2.96, HBA 10, HBD 2, PSA 141.2, QED 0.4
Compound (SMILES): Cc1noc(C)c1-c1cnc2c(c1)c(CC1CCN(S(=O)(=O)c3ccccc3)CC1)cn2C
InChIKey: HUBRWEBSQQIJLL-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Alphascreen Binding Assay: Binding of compounds of formula (I) with bromodomain 2, 3 4 was assessed using Alphascreen binding assay. The inhibition of the interaction between bromodomain and its acetylated target protein (Filippakopoulos P et al. 2012) was measured quantitatively using recombinant BRD proteins, an acetylated Histone 4 peptide and AlphaScreen technology. In absence of inhibition the BRD protein bound to AlphaScreen nickel chelate acceptor beads can interact with the acetylated Histone 4 peptide which is immobilized by the AlphaScreen Streptavidin coated beads. This interaction brings donor and acceptor beads in proximity. The close proximity allows the singlet oxygen produced by laser excitation of the donor beads to reach the acceptor beads and generate a luminescence signal. BRD inhibitors result in a decrease in the proximity signal through an inhibition of the BRD-acetylated peptide interaction.Recombinant human bromodomains containing the N-terminal bromodomain (BRD2-BD1 (71-194), BRD3-BD1 (24-144) and BRD4-BD1 (44-164)) or dual bromodomains (BRD4-BD12 (1-477), BRD4-BD12 (1-472)) were prepared and purified as described in protein expression and purification session.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 464.59, ALogP 4.49, HBA 6, HBD 0, PSA 81.23, QED 0.43
Compound (SMILES): COc1cnc([C@@H](C)[C@H](C)S(=O)(=O)Nc2nnc(-c3cccc(OC)n3)n2C2CC2)nc1
InChIKey: YQLZLMDHUUICMU-STQMWFEESA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in 9 μL assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl and 0.1% (w/v) BSA], 1 μL of diluted test compound (starting with 0.75 mM, 2-fold serial dilution with DMSO, total 22 points), 10 μL of 18 μM GDP (final concentration of 3 μM GDP), 20 μL of 0.25 μg/mL membrane protein expressing human APJ receptor captured with WGA PS beads (final concentration of 5 μg per well), and 20 μL of 0.3 nM [35S]GTPγS (final concentration is 0.1 nM [35S]GTPγS)(Perkin Elmer Life and Analytical Sciences, Waltham USA). One column of the plate was 1 μL of DMSO as background and another column of the plate was 1 μL of 180 μM Pyr-Apelin-13 which was used as control at a final concentration of 3 μM. Incubation was at RT for 90 min and the microplate was read using a ViewLux ultra HTS Microplate Imager.
Measurement: EC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 459.53, ALogP 2.42, HBA 10, HBD 1, PSA 134.01, QED 0.51
Compound (SMILES): CCn1c(=O)c2cc(S(=O)(=O)NC3(C)CC3)ccc2n(Cc2ccccc2)c1=O
InChIKey: RZVNHRFNGGKTCH-UHFFFAOYSA-N
Target: Poly(ADP-ribose) glycohydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: Q86W56
Assay: PARG Assay: PARG In vitro assays were conducted in a total volume of 15 ul in a standard 384 well format. 5 ul of Human Full Length PARG (Produced internally by Astra Zeneca), used at a final reaction concentration of 80 pM, was added to 5 ul of Ribosylated PARP substrate (also produced internally by Astra Zeneca) at final reaction concentration of 4.5 nM in assay buffer (50 mM Tris pH7.4, 0.1 mg/ml BSA, 3 mM EDTA, 0.4 mM EGTA, 1 mM DTT, 0.01% Tween 20, 50 mM KCl). The reaction was incubated at room temperature for 10 minutes and then 5 ul detection reagent was added. Detection Reagent consists of 42 nM MAb Anti-6HIS XL665 (CisBio: 61HISXLB) and 2.25 nM Streptavidin Europium Cryptate (CisBio: 610SAKLB), both at 3× working stock concentrations (final concentrations of 14 nM and 0.75 nM respectively), in a detection buffer of 50 mM Tris pH7.4, BSA at 0.1 mg/ml and KF at 100 mM. Following incubation at room temperature for 60 minutes in the dark, TR-FRET signal was measured at Ex 340 and Em 665 and Em 620. A ratio was calculated as Em665/EM620×104 for each well and used to calculate percent inhibition for test compounds.
Measurement: IC50 = 2420 nM (pChEMBL 5.619999885559082)
Descriptors: MW 413.5, ALogP 2.06, HBA 6, HBD 1, PSA 90.17, QED 0.67
Compound (SMILES): CN1C(=O)CCc2c1ccc(-c1n[nH]cc1CC1CCN(C(=O)c3cnn(C)c3)CC1)c2F
InChIKey: YZJBSCRWRHBKRY-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Inhibition Assay: The expression plasmids contains the ORF for either human/cyno CYP11B1 or CYP11B2 under the control of a suitable promoter (CMV-promoter) and a suitable resistance marker (neomycin). Using standard techniques the expression plasmid is transfected into G-402 cells and these cells are then selected for expressing the given resistance markers. Individual cell-clones are then selected and assessed for displaying the desired enzymatic activity using 11-Deoxycorticosterone (Cyp11B2) or 11-Deoxycortisol (Cyp11B1) as a substrate.G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 μg/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 0.723 nM (pChEMBL 9.140000343322754)
Descriptors: MW 450.52, ALogP 2.95, HBA 5, HBD 1, PSA 87.12, QED 0.66
Compound (SMILES): CN1C(=O)[C@@H](NC(=O)c2nc(C#N)c(Cc3cccc(C#N)c3)s2)COc2cccnc21
InChIKey: NUTUZOGGADALCH-INIZCTEOSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Fluorescent Polarization Binding (FP Binding) assay: Test compounds were diluted in DMSO and 0.1 μL of solution was dispensed to each well of a 384-well white solid microplate. The assay buffer was 50 mM HEPES pH 7.5, 50 mM NaCl, 30 mM MgCl2. The buffer was supplemented with 0.02% CHAPS, 0.01% of Pluronic F127, 0.1 mg/mL BSA and 1 mM DTT. MnCl2, 5 mM, was included in the assay buffer on the day of the experiment. The enzymatic reaction comprised 1.5 μg/mL GST-hRIPK1 (8-327) and 50 μM ATP for receptor interacting protein kinase 1 and 15 μM ATP. 5 μL of enzyme and 5 μL of ATP were added to the plate at twice the final assay concentration and incubated at room temperature for 3 hours. Following this reaction, 10 μL of ADP-Glo reagent (Promega) was added to each well and incubated for 40 min at room temperature. This stops the kinase reaction and depletes any remaining ATP. 20 μL of ADP-Glo detection reagent was then added to each well and incubated at room temperature for at least 15 minutes. The detection reagent converts ADP to ATP and introduces luciferase and luciferin to detect ATP. The luminescence is then measured with the Envision (PerkinElmer) plate reader. Test compound inhibition was expressed as percent inhibition of internal assay controls. For concentration response curves, normalized data is fit and IC50 determined using XL-fit (IDBS) for Excel. The IC50 were averaged to determine a mean value, for a minimum of two independent experiments.
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 444.48, ALogP 2.03, HBA 8, HBD 1, PSA 132, QED 0.65
Compound (SMILES): O=C1c2cc(CNc3ccccc3Cl)nn2CCN1c1ccc(F)cc1
InChIKey: AKEOPJHLAPVGQC-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P31424
Assay: mGlu3 Ca2+ Flux Assay: Gα15/TREx cells stably expressing rat mGlu3 were plated in black-walled, clear-bottomed, poly-D-lysine coated 384-well plates in 20 μL of assay medium (DMEM containing 10% dialyzed FBS, 20 mM HEPES, 25 ng/mL tetracycline, 100 units/mL penicillin/streptomycin plus 250 ng/mL Fungizone, and 1 mM sodium pyruvate) at a density of 15K cells/well. The cells were grown overnight at 37° C. in the presence of 5% CO2. The next day, medium was removed and the cells incubated with 20 μL of 2.3 μM Fluo-4, AM prepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10% (w/v) pluronic acid F-127 and diluted in assay buffer (Hank's balanced salt solution, 20 mM HEPES, and 2.5 mM probenecid) for 60 minutes at room temperature. Dye was removed, 20 μL of assay buffer was added, and the plate was incubated for 10 minutes at room temperature.
Measurement: IC50 = 2190 nM (pChEMBL 5.659999847412109)
Descriptors: MW 370.82, ALogP 3.95, HBA 4, HBD 1, PSA 50.16, QED 0.76