lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C/C(C/C=C/c1ccccc1)=C1/O[C@@H]2CC(=O)N2C1C(=O)OCc1ccccc1
InChIKey: NMQXSANBHIOUJJ-MHIZXGSHSA-N
Target: Beta-lactamase TEM (SINGLE PROTEIN), Bacteria
UniProt: P62593
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 89 nM (pChEMBL 7.050000190734863)
Descriptors: MW 403.43, ALogP 3.24, HBA 5, HBD 0, PSA 72.91, QED 0.31
Compound (SMILES): CC(=O)NC/C=C1\CCc2ccc3nc(CCCCc4ccccc4)oc3c21
InChIKey: HEWLIENXMUSPDG-HMMYKYKNSA-N
Target: Melatonin receptor type 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P48039
Assay: Binding Assay: Binding assay using melatonin receptors 1 or 2.
Measurement: IC50 = 0.079 nM (pChEMBL 10.100000381469727)
Descriptors: MW 374.48, ALogP 4.86, HBA 3, HBD 1, PSA 55.13, QED 0.6
Compound (SMILES): O=C(O)/C(C/C=C/CN(Cc1ccccc1)C(=O)OCc1ccccc1)=C1/O[C@@H]2CC(=O)N2C1C(=O)OCc1ccccc1
InChIKey: HXKNILWLBVUKER-PGNJWVLTSA-N
Target: Beta-lactamase SHV-1 (SINGLE PROTEIN), Escherichia coli
UniProt: P0AD63
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 596.64, ALogP 4.81, HBA 7, HBD 1, PSA 122.68, QED 0.14
Compound (SMILES): C=CC/C(C(=O)O)=C1\O[C@@H]2CC(=O)N2C1C(=O)OCc1ccccc1
InChIKey: RBWUPNXZJVLGSA-BSXLFBHHSA-N
Target: Beta-lactamase SHV-1 (SINGLE PROTEIN), Escherichia coli
UniProt: P0AD63
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 62.2 nM (pChEMBL 7.210000038146973)
Descriptors: MW 343.34, ALogP 1.6, HBA 5, HBD 1, PSA 93.14, QED 0.36
Compound (SMILES): O=C/C(C/C=C/CO)=C1/O[C@@H]2CC(=O)N2C1C(=O)OCc1ccccc1
InChIKey: PIPGAOFSHNWTAH-ABDVADSRSA-N
Target: Beta-lactamase SHV-1 (SINGLE PROTEIN), Escherichia coli
UniProt: P0AD63
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 216.9 nM (pChEMBL 6.659999847412109)
Descriptors: MW 357.36, ALogP 1.08, HBA 6, HBD 1, PSA 93.14, QED 0.26
Compound (SMILES): NC(=S)N/N=C(/c1ccccc1)c1cccc(C(O)c2ccccc2)c1
InChIKey: FJWDUFMZXRQPDL-NMWGTECJSA-N
Target: Cathepsin B (SINGLE PROTEIN), Homo sapiens
UniProt: P07858
Assay: Biological Assay: Human liver Cathepsin B (Calbiochem) was pre-incubated with test compound at various concentrations for 5 minutes at 37C. The assay was initiated by addition of substrate Z-Arg-Arg-aminomethylcoumarin (Z-R-R-AMC Bacchem) and the final assay conditions were 1.1 nM cathepsin B, 60 uM Z-R-R-AMC, 126 mM sodium potassium phosphate pH 6.0 (Fisher), 0.3 mM EDTA (Omnipure), 2.7 mM DTT (Omnipure), 0.004% BRIJ 35 (Sigma), and 2.0% DMSO (Acros) in a final volume of 200 uL. Test compounds were serially diluted with DMSO and 0.01% BRIJ 35 to include a final concentration range of 20 uM to 10 pM. The reaction was monitored fluorometrically for 5 minutes at 37C. using black 96-well Corning 3686 assay microplates with a Thermo Fluoroskan Ascent FL microplate reader at excitation and emission filter wavelengths of 355 nm and 460 nm, respectively.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 361.47, ALogP 3.35, HBA 3, HBD 3, PSA 70.64, QED 0.37
Compound (SMILES): C=CC/C(C(=O)O)=C1\O[C@@H]2CC(=O)N2C1C(=O)OC
InChIKey: DMUDPEVSVHCUNB-KZELUKOJSA-N
Target: Beta-lactamase SHV-1 (SINGLE PROTEIN), Escherichia coli
UniProt: P0AD63
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 339 nM (pChEMBL 6.46999979019165)
Descriptors: MW 267.24, ALogP 0.03, HBA 5, HBD 1, PSA 93.14, QED 0.33
Compound (SMILES): CCC/C(C(=O)O)=C1\O[C@@H]2CC(=O)N2C1C(=O)O
InChIKey: QAABSWVJCGWPMP-SKQQWWGDSA-N
Target: Beta-lactamase TEM (SINGLE PROTEIN), Bacteria
UniProt: P62593
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 254 nM (pChEMBL 6.590000152587891)
Descriptors: MW 255.23, ALogP 0.17, HBA 4, HBD 2, PSA 104.14, QED 0.55
Compound (SMILES): O=C(O)/C=C1\O[C@@H]2CC(=O)N2C1(C/C=C/c1ccccc1)C(=O)OCc1ccccc1
InChIKey: TXPJIXCYUFODBL-WWTCDDSASA-N
Target: Beta-lactamase SHV-1 (SINGLE PROTEIN), Escherichia coli
UniProt: P0AD63
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 624.3 nM (pChEMBL 6.210000038146973)
Descriptors: MW 419.43, ALogP 3.13, HBA 5, HBD 1, PSA 93.14, QED 0.42
Compound (SMILES): CC(C)(C)/C=C(\C#N)C(=O)N1CCCC(n2nc(-c3ccc(Oc4ccccc4)cc3)c3c(N)ncnc32)C1
InChIKey: BRHJRWOQJXUVRF-LTGZKZEYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Enzymatic Activity Assay: A Caliper-based kinase assay (Caliper Life Sciences, Hopkinton, Mass.) was used to measure inhibition of Btk kinase activity of a compound of Formula (IA), (I′) or (I). Serial dilutions of test compounds were incubated with human recombinant Btk (2 nM), ATP (40 μM) and a phosphoacceptor peptide substrate FAM-GEEPLYWSFPAKKK-NH2 (1 μM) at room temperature for 3 h. The reaction was then terminated with EDTA, final concentration 20 mM and the phosphorylated reaction product was quantified on a Caliper Desktop Profiler (Caliper LabChip 3000). Percent inhibition was calculated for each compound dilution and the concentration that produced 50% inhibition was calculated.
Measurement: IC50 = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 521.63, ALogP 5.53, HBA 8, HBD 1, PSA 122.95, QED 0.27
Compound (SMILES): CC(C)(C)/C=C(\C#N)C(=O)N1CCC[C@@H](n2nc(-c3ccc(Oc4ccccc4)cc3F)c3c(N)ncnc32)C1
InChIKey: KZMQPYCXSAGLTB-ZWUNQBBJSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition of BTK (unknown origin) using peptide substrate in presence of ATP by caliper electrophoresis method
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 539.62, ALogP 5.67, HBA 8, HBD 1, PSA 122.95, QED 0.26
Compound (SMILES): CC(C)=C[C@@H]1CN(C(=O)[C@H](CC(C)C)NC(=O)c2ccc(-c3nc(N4CCN(C)CC4)sc3F)cc2)C2C(=O)COC21
InChIKey: UTDIUZBVSSNGOU-JIRKYJOESA-N
Target: Cathepsin S (SINGLE PROTEIN), Homo sapiens
UniProt: P25774
Assay: Biological Assay: Standard assay conditions for the determination of kinetic constants used a fluorogenic peptide substrate, typically H-D-Ala-Leu-Lys-AMC, and were determined in either 100 mM Mes/Tris, pH 7.0 containing 1 mM EDTA and 10 mM 2-mercaptoethanol or 100 mMNa phosphate, imM EDTA, 0.1% PEG4000 pH 6.5 or 100 mM Na acetate, pH 5.5 containing 5 mM EDTA and 20 mM cysteine, in each case optionally with 1M DTT as stabiliser. The enzyme concentration used was 5 nM. The stock substrate solution was prepared at 10 mM in DMSO. Screens were carried out at a fixed substrate concentration of 60 μM and detailed kinetic studies with doubling dilutions of substrate from 250 μM. The total DMSO concentration in the assay was kept below 3%. All assays were conducted at ambient temperature. Product fluorescence (excitation at 390 nm, emission at 460 nm) was monitored with a Labsystems Fluoroskan Ascent fluorescent plate reader.
Measurement: Ki = 14500 nM (pChEMBL 4.840000152587891)
Descriptors: MW 597.76, ALogP 3.61, HBA 8, HBD 1, PSA 95.08, QED 0.46
Compound (SMILES): CCC/C(C(=O)O)=C1\O[C@@H]2CC(=O)N2C1C(=O)OC
InChIKey: ALXADSUETYDFDB-KZELUKOJSA-N
Target: Beta-lactamase SHV-1 (SINGLE PROTEIN), Escherichia coli
UniProt: P0AD63
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 294.6 nM (pChEMBL 6.53000020980835)
Descriptors: MW 269.25, ALogP 0.26, HBA 5, HBD 1, PSA 93.14, QED 0.45
Compound (SMILES): C=CCC1(C(=O)OCc2ccccc2)/C(=C/CO)O[C@@H]2CC(=O)N21
InChIKey: ADMWSBHYYMUDFV-XKQBMVGDSA-N
Target: Beta-lactamase TEM (SINGLE PROTEIN), Bacteria
UniProt: P62593
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 23621.5 nM (pChEMBL 4.630000114440918)
Descriptors: MW 329.35, ALogP 1.51, HBA 5, HBD 1, PSA 76.07, QED 0.49
Compound (SMILES): C=CC/C(C=O)=C1\O[C@@H]2CC(=O)N2C1C(=O)OC
InChIKey: HLBYYYVPQDVZIQ-XRRNHPNGSA-N
Target: Beta-lactamase SHV-1 (SINGLE PROTEIN), Escherichia coli
UniProt: P0AD63
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 80.5 nM (pChEMBL 7.090000152587891)
Descriptors: MW 251.24, ALogP 0.15, HBA 5, HBD 0, PSA 72.91, QED 0.23
Compound (SMILES): CCCC1(C(=O)O)/C(=C/CO)O[C@@H]2CC(=O)N21
InChIKey: FPJUOUJTKUOLCV-JMKIFZRZSA-N
Target: Beta-lactamase TEM (SINGLE PROTEIN), Bacteria
UniProt: P62593
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 17789 nM (pChEMBL 4.75)
Descriptors: MW 241.24, ALogP 0.07, HBA 4, HBD 2, PSA 87.07, QED 0.68
Compound (SMILES): CC(=O)OC/C=C/C/C(C=O)=C1\O[C@@H]2CC(=O)N2C1C(=O)OCc1ccccc1
InChIKey: OUNQKFHIKDLFII-JWTJEGOJSA-N
Target: Beta-lactamase TEM (SINGLE PROTEIN), Bacteria
UniProt: P62593
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 139.9 nM (pChEMBL 6.849999904632568)
Descriptors: MW 399.4, ALogP 1.65, HBA 7, HBD 0, PSA 99.21, QED 0.22
Compound (SMILES): C=CCC1(C(=O)OC)/C(=C/C=O)O[C@@H]2CC(=O)N21
InChIKey: WEJZNOFAZVDYFB-YTVFTGSSSA-N
Target: Beta-lactamase SHV-1 (SINGLE PROTEIN), Escherichia coli
UniProt: P0AD63
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 21303.5 nM (pChEMBL 4.670000076293945)
Descriptors: MW 251.24, ALogP 0.15, HBA 5, HBD 0, PSA 72.91, QED 0.23
Compound (SMILES): COC(=O)C1(C/C=C/c2ccccc2)/C(=C/C(=O)O)O[C@@H]2CC(=O)N21
InChIKey: FJEAGJSMLOQFME-MJSMECKXSA-N
Target: Beta-lactamase SHV-1 (SINGLE PROTEIN), Escherichia coli
UniProt: P0AD63
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 2307.2 nM (pChEMBL 5.639999866485596)
Descriptors: MW 343.34, ALogP 1.56, HBA 5, HBD 1, PSA 93.14, QED 0.5
Compound (SMILES): NC(=O)c1cccc2c(NCC3CC=C/C(=N/C(O)c4ccn[nH]4)C3)ncnc12
InChIKey: WHOKGFKDKLABFY-ZMFRSBBQSA-N
Target: Aurora kinase B (SINGLE PROTEIN), Homo sapiens
UniProt: Q96GD4
Assay: Enzyme Assay: The Aurora assays described here are performed on two Caliper Life Sciences systems: the LC3000 and the Desktop Profiler. These provide data on enzyme activity via measurement of the relative amounts of phosphorylated or unphosphorylated fluorescently labelled substrate peptide at the end of an enzymatic reaction. These different states of peptide are resolved by applying a potential difference across the sample. The presence of the charged phosphate group on the product (as opposed to the substrate) causes a different peptide mobility between the two peptides. This is visualized by excitation of the fluorescent label on the substrate and product peptides and represented as peaks within the analysis software.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 391.44, ALogP 1.96, HBA 7, HBD 4, PSA 142.17, QED 0.51
Compound (SMILES): O=C1OC(CI)C/C1=C1/O[C@@H]2CC(=O)N2C1C(=O)OCc1ccccc1
InChIKey: ROBNSNBYRBBNOO-RZFORLEUSA-N
Target: Beta-lactamase SHV-1 (SINGLE PROTEIN), Escherichia coli
UniProt: P0AD63
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 469.23, ALogP 1.69, HBA 6, HBD 0, PSA 82.14, QED 0.22
Compound (SMILES): CC(=O)OC/C=C/CC1(C(=O)OCc2ccccc2)/C(=C/C=O)O[C@@H]2CC(=O)N21
InChIKey: BGQRDDSRQMAZIR-VOXXZNBISA-N
Target: Beta-lactamase (SINGLE PROTEIN), Escherichia coli K-12
UniProt: P00811
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 29396 nM (pChEMBL 4.53000020980835)
Descriptors: MW 399.4, ALogP 1.65, HBA 7, HBD 0, PSA 99.21, QED 0.22
Compound (SMILES): C=CCC1(C(=O)OCc2ccccc2)/C(=C/C=O)O[C@@H]2CC(=O)N21
InChIKey: SPTMQRZXEWVYFQ-XKQBMVGDSA-N
Target: Beta-lactamase TEM (SINGLE PROTEIN), Bacteria
UniProt: P62593
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 945.4 nM (pChEMBL 6.019999980926514)
Descriptors: MW 327.34, ALogP 1.72, HBA 5, HBD 0, PSA 72.91, QED 0.26
Compound (SMILES): NC(=O)c1cccc2c(NCC3CC=C/C(=N/C(O)c4c[nH]c5ncccc45)C3)ncnc12
InChIKey: MVVLRGOQAYKEMN-BVMLUPFRSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Enzyme Assay: The Aurora assays described here are performed on two Caliper Life Sciences systems: the LC3000 and the Desktop Profiler. These provide data on enzyme activity via measurement of the relative amounts of phosphorylated or unphosphorylated fluorescently labelled substrate peptide at the end of an enzymatic reaction. These different states of peptide are resolved by applying a potential difference across the sample. The presence of the charged phosphate group on the product (as opposed to the substrate) causes a different peptide mobility between the two peptides. This is visualized by excitation of the fluorescent label on the substrate and product peptides and represented as peaks within the analysis software.
Measurement: IC50 = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 441.5, ALogP 3.12, HBA 7, HBD 4, PSA 142.17, QED 0.36
Compound (SMILES): O=C(O)/C(C/C=C/c1ccccc1)=C1/O[C@@H]2CC(=O)N2C1C(=O)OCc1ccccc1
InChIKey: FVJOWYDVEXFNKI-FWHGTPTCSA-N
Target: Beta-lactamase SHV-1 (SINGLE PROTEIN), Escherichia coli
UniProt: P0AD63
Assay: Cell Free Inhibition Assay: The inhibitory concentrations (IC50, [uM]) of the beta -lactamase inhibitors against purified TEM-1, SHV-1 and AmpC beta -lactamases are assessed by determining the concentration of inhibitor at which 50% of the nitrocefin hydrolysis is inhibited by the particular enzyme. Assays are performed with beta -lactamases expressed in the pET system (Novagen, San Diego, Calif.) without signal peptides. They contain an N-terminal hexa-Histidine tag that is used for purification on Ni-NTA (Qiagen, Hilden, Germany). The compounds are prepared as 50 mM stocks in DMSO and diluted into buffer P1 (50 mM phosphate, pH 7) to a final concentration of 10% DMSO. All further dilutions are done in P2 (P1 with 10% DMSO). Enzyme and compound dilutions are pre-incubated for 10 min at 37 C. and the reaction is started with the addition of pre-warmed (37 C.) nitrocefin at a final concentration of 50 mM. The change in absorption at 490 nm is followed at 37 C. for 10 min with 30 s intervals.
Measurement: IC50 = 36.3 nM (pChEMBL 7.440000057220459)
Descriptors: MW 419.43, ALogP 3.13, HBA 5, HBD 1, PSA 93.14, QED 0.42
Compound (SMILES): CCOn1c(N[C@H](C)c2ccc(C(F)(F)F)cc2)nc2c(c1=O)CN(C(=O)c1ccc(C#N)cc1F)CC2
InChIKey: TUNWVDSPXXVZAA-OAHLLOKOSA-N
Target: Prolactin-releasing peptide receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P49683
Assay: Intracellular Calcium Mobilization Assay: HEK293 cells expressing human GPR10 were maintained in Dulbecco's Modified Eagles' medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 20 mM HEPES at 37° C. in a 5% CO2 incubator. GPR10-expressing HEK293 cells were placed in poly-D-lysine coated 96-well culture plates and cultured for 18-24 hr before the test at a density of 3×104 cells/well. The cells were incubated with 2.5 nM Fluo-8 for 1 hr at room temperature in Recording Buffer containing 1% bovine serum albumin, 0.01% pluronic F-127 and 20 mM HEPES, Hanks Balanced Buffered Saline, pH 7.4. The cells were incubated with test compounds for 15 min at room temperature, and then PrRP (the final concentration, 1 nM) was added into the medium. The changes in intracellular calcium-dependent fluorescence were monitored using a fluorescence imaging plate reader (FDSS3000, Hamamatsu Photonics K.K.). Fluo-8 fluorescence was measured with excitation at 490 nm and emission at 520 nm.
Measurement: IC50 = 227 nM (pChEMBL 6.639999866485596)
Descriptors: MW 529.49, ALogP 4.09, HBA 7, HBD 1, PSA 100.25, QED 0.48
Compound (SMILES): C[C@@]1(C#N)CS(=O)(=O)C[C@H]1n1cc(C(N)=O)c(Nc2ccc(F)cc2)n1
InChIKey: QPHALRREIYHCHW-CZUORRHYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at ambient temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at ambient temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at ambient temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 377.4, ALogP 1.36, HBA 7, HBD 2, PSA 130.87, QED 0.83
Compound (SMILES): CNC(=O)[C@@H](NC(=O)c1cc(OCC(F)(F)F)c(Cl)cn1)C(C)(C)C
InChIKey: WDYJUOUECDTFRD-LLVKDONJSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 381.78, ALogP 2.57, HBA 4, HBD 2, PSA 80.32, QED 0.82
Compound (SMILES): CCC(=O)N[C@@H]1c2cncc(-c3cc4c(cc3F)N(C)C(=O)CC4)c2CC[C@H]1F
InChIKey: WKHNTIGVBRUGAV-VGOFRKELSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Cellular Enzyme 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 μM 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 = 2389.7 nM (pChEMBL 5.619999885559082)
Descriptors: MW 399.44, ALogP 3.65, HBA 3, HBD 1, PSA 62.3, QED 0.86
Compound (SMILES): Cc1nc(C(C)Nc2ncc(F)c(N3C(=O)OC[C@@H]3[C@@H](C)O)n2)oc1-c1ccc(Cl)cc1
InChIKey: HLHUIGPXIQCWLI-ZLXOECBPSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: LC-MS Detection of 2-HG Assay: The biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG. The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below). LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 461.88, ALogP 4.11, HBA 8, HBD 2, PSA 113.61, QED 0.56
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2[C@H](C)CO[C@H]2Cn2cc(-c3ccc(F)cn3)cn2)c1
InChIKey: HROJDHGISWMAPH-ZHRRBRCNSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Antagonistic Activity Assay: The antagonistic activities of the test compounds on human orexin-1 receptor (hOX1R) and orexin-2 receptor (hOX2R) were measured by modifying from the method described in literature (Toshikatsu Okumura et al., Biochemical and Biophysical Research Communications 280, 976-981, 2001). Chinese hamster ovary (CHO) cells forcibly expressing the hOX1R and hOX2R were seeded into a 96 well Black clear bottom plate (Nunc) at 20,000 cells per well, which were cultured in Ham's F-12 medium containing 0.1 mM MEM non-essential amino acids, 0.5 mg/ml G418, 10% fetal bovine serum (all by Invitrogen) for 16 hours under the conditions of 37° C., 5% CO2. After removing the medium, 100 uL of 0.5 uM Fluo-4AM ester (Dojin) in an assay buffer (25 mM HEPES (Dojin), Hank's balanced salt solution (Invitrogen), 0.1% bovine serum albumin, 2.5 mM probenecid, 200 ug/ml Amaranth (all by Sigma-Aldrich), pH 7.4) was added and the cells were incubated for 60 minutes at 37° C., 5% CO2. After removing the medium, 100 uL of 0.5 uM Fluo-4AM ester (Dojin) in an assay buffer (25 mM HEPES (Dojin), Hank's balanced salt solution (Invitrogen), 0.1% bovine serum albumin, 2.5 mM probenecid, 200 ug/ml Amaranth (all by Sigma-Aldrich), pH 7.4) was added and the cells were incubated for 60 minutes at 37° C., 5% CO2. After removing the assay buffer containing fluo-3AM ester, the test compound was dissolved in dimethyl sulfoxide to be 10 mM and diluted with the assay buffer, 150 uL of which was added and incubated for 30 minutes.
Measurement: IC50 = 460.1 nM (pChEMBL 6.340000152587891)
Descriptors: MW 447.47, ALogP 2.86, HBA 8, HBD 0, PSA 90.96, QED 0.47
Compound (SMILES): COc1cccc(-n2cc(C(=O)Nc3ccc([C@H]4CNCCO4)cc3)nn2)c1
InChIKey: SGMXMLCODCILQQ-LJQANCHMSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding (rat TAAR1): HEK-293 cells stably expressing rat TAAR1 were maintained at 37Â° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56Â° C.), penicillin/streptomycin (1%), and 375 Î¼g/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1'000 rpm for 5 min at 4Â° C., frozen and stored at â¿¿80Â° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14'000 rpm for 20 s. The homogenate was centrifuged at 48'000Ã¿g for 30 min at 4Â° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14'000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at â¿¿80Â° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 Î¼M unlabeled ligand. All compounds were tested at a broad range of concentrations (10 Î¼M to 10 Î¼M) in duplicates. The test compounds (20 Î¼l/well) were transferred into a 96 deep well plate (TreffLab), and 180 Î¼l of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 Î¼l of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3Ã¿Kd in nM and 500 Î¼l of the membranes (resuspended at 50 Î¼g protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4Â° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 Î¼l of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 9.2 nM (pChEMBL 8.039999961853027)
Descriptors: MW 379.42, ALogP 2.19, HBA 7, HBD 2, PSA 90.3, QED 0.71
Compound (SMILES): CCOc1ccc(Cc2cc([C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)c3c(c2Cl)CCCC3)cc1
InChIKey: JSQPKDWJZHMHMC-HFBCXCLPSA-N
Target: Sodium/glucose cotransporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P13866
Assay: SGLT1 Inhibition Assay: For sodium-dependent glucose transport assay, cells expressing hSGLT1 were seeded into a 96-well culture plate at a density of 5×104 cells/well in RPMI medium 1640 containing 10% fetal bovine serum. The cells were used 1 day after plating. They were incubated in pretreatment buffer (10 mM HEPES, 5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl2, and 1 mM MgCl2, pH 7.4) at 37 °C. for 10 min. They were then incubated in uptake buffer (10 mM HEPES, 5 mM Tris, 140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM 14C-nonlabeled AMG pH 7.4) containing 14C-labeled (8 μM) and inhibitor or dimethyl sulfoxide (DMSO) vehicle at 37 °C. for 2 h. Cells were washed twice with washing buffer (pretreatment buffer containing 10 mM AMG at room temperature) and then the radioactivity was measured using a liquid scintillation counter.
Measurement: IC50 = 33.2 nM (pChEMBL 7.480000019073486)
Descriptors: MW 462.97, ALogP 2.72, HBA 6, HBD 4, PSA 99.38, QED 0.53
Compound (SMILES): N#Cc1ccc(-c2cc(C(=O)Nc3ccc(OC(F)(F)Cl)cc3)cnc2N2CC[C@@H](O)C2)[nH]1
InChIKey: JTQKMBJRQNMMGH-MRXNPFEDSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Caliper Assay: The assay plates were prepared by addition of 50 nL per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 μL per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 4 μM ATP, 4 μM peptide (FITC-Ahx-EAIYAAPFAKKK-NH2)) and 4.5 μL per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 3.5 nM ABL (ABL(64-515), produced in-house from E. coli)). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 μL per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology. Briefly, samples from terminated kinase reactions were applied to the chip. Analytes are transported through the chip by constant buffer flow and the migration of the substrate peptide is monitored by the fluorescence signal of its label. Phosphorylated peptide (product) and unphosphorylated peptide (substrate) are separated in an electric field by their charge/mass ratio.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 473.87, ALogP 3.94, HBA 6, HBD 3, PSA 114.27, QED 0.47
Compound (SMILES): COc1cc(C(=O)N2CC(C)OCC2C2CC(O)C2)cc2nc(N[C@H](CF)c3cccc(Cl)c3)oc12
InChIKey: OAZBEQQNMLSDQD-QGSYWXNBSA-N
Target: Thyroid hormone receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P10828
Assay: Inhibition of purified human plasma thrombin using Boc-Asp(OBzl)-Pro-Arg-AMC substrate incubated for 30 mins by fluorescence based assay
Measurement: IC50 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 517.99, ALogP 4.61, HBA 7, HBD 2, PSA 97.06, QED 0.47
Compound (SMILES): Nc1ncc(C2CCOCC2)cc1-c1ccc(C(=O)N[C@H](CO)c2ccccc2)cc1F
InChIKey: NCUYHPFPTDVMQR-HSZRJFAPSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: AlphaScreen Assay: Compound potency against activated ERK2 is determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay is carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration is used for generating IC50 curves. Compound dilution series are prepared in 100% DMSO. ERK2 is preincubated with compounds for 30 minutes at ambient temperature. Reaction is initiated by addition of a substrate cocktail of ERKtide peptide and ATP and is allowed to proceed for 2-3 hours at ambient temperature. Reaction is terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 ug/mL of AlphaScreen Protein A Acceptor Beads, 10 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 ug/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.).
Measurement: IC50 = 17.2 nM (pChEMBL 7.760000228881836)
Descriptors: MW 435.5, ALogP 3.83, HBA 5, HBD 3, PSA 97.47, QED 0.55