lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cc2ncccc2c(OC[C@H]2CNC(=O)C2)n1
InChIKey: LGLTUBADVDGOPY-SNVBAGLBSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Kinase Glo Luminescence Kinase Assay: Recombinant human Syk (amino acids 342-635) was expressed as a fusion protein with an N-terminal GST tag, affinity-purified and deep-frozen at a concentration of approx. 50 - 100 uM in test buffer (25 mM HEPES pH7.5; 25 mM MgCI2;5 mM MnCI2; 50 mM KCI; 0.2% BSA; 0.01 % CHAPS; 100 uM Na3V04; 0.5 mM DTT) and 10% glycerol at -80 C until use.The catalytic activity of the GST-Syk kinase fusion protein was determined using the Kinase Glo Luminescence Kinase test (Promega; V6712). In this homogeneous test the amount of ATP remaining after the kinase reaction is quantified by a luciferin-luciferase reaction using luminescence. The luminescence signal obtained correlates with the amount of ATP still present and thus correlates inversely with the activity of the protein kinase. Method:The test compounds were dissolved in 100 % DMSO at a concentration of 10 mM and diluted in DMSO to a concentration of 1 mM.
Measurement: IC50 = 5.1 nM (pChEMBL 8.289999961853027)
Descriptors: MW 257.29, ALogP 1.45, HBA 4, HBD 1, PSA 64.11, QED 0.9
Compound (SMILES): Cc1c([C@@H](O)CN2CCN(C[C@H](O)c3ccc4c(c3C)COC4=O)CC2)ccc2c1COC2=O
InChIKey: OCKGFTQIICXDQW-ZEQRLZLVSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Inhibition of ROMK (unknown origin) expressed in CHO cells after 6 mins by electrophysiological assay
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 466.53, ALogP 2.03, HBA 8, HBD 2, PSA 99.54, QED 0.62
Compound (SMILES): Cc1c([C@H](O)CN2CCN(C[C@@H](O)c3ccc4c(c3C)COC4=O)CC2)ccc2c1COC2=O
InChIKey: OCKGFTQIICXDQW-DNQXCXABSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Inhibition of ROMK (unknown origin) after 30 mins by 86Rb+ efflux assay
Measurement: IC50 = 134 nM (pChEMBL 6.869999885559082)
Descriptors: MW 466.53, ALogP 2.03, HBA 8, HBD 2, PSA 99.54, QED 0.62
Compound (SMILES): Cc1c([C@H](O)CN2CCN(C[C@H](O)c3ccc4c(c3C)COC4=O)CC2)ccc2c1COC2=O
InChIKey: OCKGFTQIICXDQW-PSWAGMNNSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Inhibition of ROMK (unknown origin) after 30 mins by 86Rb+ efflux assay
Measurement: IC50 = 112 nM (pChEMBL 6.949999809265137)
Descriptors: MW 466.53, ALogP 2.03, HBA 8, HBD 2, PSA 99.54, QED 0.62
Compound (SMILES): C[C@H]1Cn2cc(C(N)=O)nc2-c2cc(C#CC(C)(C)O)ccc2O1
InChIKey: FCLUEVDLZSWFCZ-NSHDSACASA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/untagged recombinant full length human p85alpha expressed in baculovirus infected Sf21 insect cells using PIP2:3PS lipid substrate after 120 mins by ADP-Glo assay
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 325.37, ALogP 1.55, HBA 5, HBD 2, PSA 90.37, QED 0.78
Compound (SMILES): C[C@@H]1Cn2cc(C(N)=O)nc2-c2cc(C#CC(C)(C)O)ccc2O1
InChIKey: FCLUEVDLZSWFCZ-LLVKDONJSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5'-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5'-diphosphate) assay (BellBrook Labs). Purified NIK (0.2-1 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl.sub.2, 2 mM dithiothreitol, 10 uM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 12 ug/mL ADP antibody and 4 nM ADP-AlexaFluor.RTM. 633 tracer. Reactions were quenched by the addition of 20 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation.
Measurement: Ki = 620 nM (pChEMBL 6.210000038146973)
Descriptors: MW 325.37, ALogP 1.55, HBA 5, HBD 2, PSA 90.37, QED 0.78
Compound (SMILES): COC[C@@](C)(O)C#Cc1cc2c(cc1F)C1CC(C1)n1c-2nc(C(N)=O)c1[C@H](O)c1ccnn1C
InChIKey: CEDFAOFDDPOULD-IXPZBHSUSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5'-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5'-diphosphate) assay (BellBrook Labs). Purified NIK (0.2-1 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl.sub.2, 2 mM dithiothreitol, 10 uM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 12 ug/mL ADP antibody and 4 nM ADP-AlexaFluor.RTM. 633 tracer. Reactions were quenched by the addition of 20 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation.
Measurement: Ki = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 479.51, ALogP 1.78, HBA 8, HBD 3, PSA 128.42, QED 0.48
Compound (SMILES): COC[C@](C)(O)C#Cc1cc2c(cc1F)C1CC(C1)n1c-2nc(C(N)=O)c1[C@H](O)c1ccnn1C
InChIKey: CEDFAOFDDPOULD-MWSNSZIRSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5'-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5'-diphosphate) assay (BellBrook Labs). Purified NIK (0.2-1 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl.sub.2, 2 mM dithiothreitol, 10 uM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 12 ug/mL ADP antibody and 4 nM ADP-AlexaFluor.RTM. 633 tracer. Reactions were quenched by the addition of 20 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation.
Measurement: Ki = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 479.51, ALogP 1.78, HBA 8, HBD 3, PSA 128.42, QED 0.48
Compound (SMILES): CN1CC[C@@](O)(C#Cc2cc3c(cc2F)C2CC(C2)n2c-3nc(C(N)=O)c2[C@H](O)c2ccnn2C)C1=O
InChIKey: AZAFMASCHOZXJQ-WUUBFYFJSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5'-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5'-diphosphate) assay (BellBrook Labs). Purified NIK (0.2-1 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl.sub.2, 2 mM dithiothreitol, 10 uM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 12 ug/mL ADP antibody and 4 nM ADP-AlexaFluor.RTM. 633 tracer. Reactions were quenched by the addition of 20 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation.
Measurement: Ki > 1200 nM (pChEMBL 5.921000003814697)
Descriptors: MW 504.52, ALogP 0.98, HBA 8, HBD 3, PSA 139.5, QED 0.45
Compound (SMILES): CN1CC[C@@](O)(C#Cc2cc3c(cc2F)C2CC(C2)n2c-3nc(C(N)=O)c2[C@@H](O)c2ccnn2C)C1=O
InChIKey: AZAFMASCHOZXJQ-OTNPQCEHSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5'-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5'-diphosphate) assay (BellBrook Labs). Purified NIK (0.2-1 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl.sub.2, 2 mM dithiothreitol, 10 uM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 12 ug/mL ADP antibody and 4 nM ADP-AlexaFluor.RTM. 633 tracer. Reactions were quenched by the addition of 20 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation.
Measurement: Ki > 1200 nM (pChEMBL 5.921000003814697)
Descriptors: MW 504.52, ALogP 0.98, HBA 8, HBD 3, PSA 139.5, QED 0.45
Compound (SMILES): CN1CC[C@](O)(C#Cc2cc3c(cc2F)C2CC(C2)n2c-3nc(C(N)=O)c2[C@H](O)c2ccnn2C)C1=O
InChIKey: AZAFMASCHOZXJQ-IFHKZOIYSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5'-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5'-diphosphate) assay (BellBrook Labs). Purified NIK (0.2-1 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl.sub.2, 2 mM dithiothreitol, 10 uM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 12 ug/mL ADP antibody and 4 nM ADP-AlexaFluor.RTM. 633 tracer. Reactions were quenched by the addition of 20 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation.
Measurement: Ki > 1200 nM (pChEMBL 5.921000003814697)
Descriptors: MW 504.52, ALogP 0.98, HBA 8, HBD 3, PSA 139.5, QED 0.45
Compound (SMILES): CN1CC[C@](O)(C#Cc2cc3c(cc2F)C2CC(C2)n2c-3nc(C(N)=O)c2[C@@H](O)c2ccnn2C)C1=O
InChIKey: AZAFMASCHOZXJQ-AJDSKFFFSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5'-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5'-diphosphate) assay (BellBrook Labs). Purified NIK (0.2-1 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl.sub.2, 2 mM dithiothreitol, 10 uM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 12 ug/mL ADP antibody and 4 nM ADP-AlexaFluor.RTM. 633 tracer. Reactions were quenched by the addition of 20 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation.
Measurement: Ki > 1200 nM (pChEMBL 5.921000003814697)
Descriptors: MW 504.52, ALogP 0.98, HBA 8, HBD 3, PSA 139.5, QED 0.45
Compound (SMILES): COC[C@](C)(O)C#Cc1cc2c(cc1F)C1CC(C1)n1c-2nc(C(N)=O)c1[C@@H](O)c1ccnn1C
InChIKey: CEDFAOFDDPOULD-KKKMIPKYSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5'-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5'-diphosphate) assay (BellBrook Labs). Purified NIK (0.2-1 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl.sub.2, 2 mM dithiothreitol, 10 uM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 12 ug/mL ADP antibody and 4 nM ADP-AlexaFluor.RTM. 633 tracer. Reactions were quenched by the addition of 20 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation.
Measurement: Ki = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 479.51, ALogP 1.78, HBA 8, HBD 3, PSA 128.42, QED 0.48
Compound (SMILES): COC[C@@](C)(O)C#Cc1cc2c(cc1F)C1CC(C1)n1c-2nc(C(N)=O)c1[C@@H](O)c1ccnn1C
InChIKey: CEDFAOFDDPOULD-OMBFLKOESA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5'-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5'-diphosphate) assay (BellBrook Labs). Purified NIK (0.2-1 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl.sub.2, 2 mM dithiothreitol, 10 uM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 12 ug/mL ADP antibody and 4 nM ADP-AlexaFluor.RTM. 633 tracer. Reactions were quenched by the addition of 20 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation.
Measurement: Ki = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 479.51, ALogP 1.78, HBA 8, HBD 3, PSA 128.42, QED 0.48
Compound (SMILES): CCCNc1cnn(C2CC(C)(C)CC(C)(C)C2)c(=O)c1Cl
InChIKey: YMCXJOJJPKTDCH-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Radioligand Binding Assay: Plasma membranes prepared from HEK 293 EBNA cells transfected with human CB1 or CB2 cannabinoid receptors (3.7 or 3.3 .mu.mol/mg protein, receptor concentration; PerkinElmer) were used for the radioligand binding studies. The composition of incubation buffer for CB1 assay was 50 mM Tris base, 2.5 mM EDTA, 5 mM MgCl.sub.2 and 0.5 mg/ml fatty acid free BSA and for CB2 assay was 50 mM Tris base, 2.5 mM EGTA, 5 mM MgCl.sub.2 and 1 mg/ml fatty acid free BSA; pH was adjusted to 7.4 by adding 1N HCl. Plasma membranes were diluted with incubation buffer to provide a final protein concentration of 2.4 .mu.g (CB1) or 8 .mu.g (CB2) per well in non-binding surface polystyrene 96-well assay plates (Corning). Compound solutions were prepared in silanized glass tubes and dispensed using pipette tips with SUPERSLIK.TM. surface. Competition studies were performed using a final concentration of 0.72 nM [.sup.3H]-CP 55,940 (100-180 Ci/mmol, specific activity; PerkinElmer) against test compounds.
Measurement: IC50 = 1023 nM (pChEMBL 5.989999771118164)
Descriptors: MW 325.88, ALogP 4.5, HBA 4, HBD 1, PSA 46.92, QED 0.89
Compound (SMILES): CC1(C)CC(n2ncc(NCCN3CCOCC3)c(Br)c2=O)CC(C)(C)C1
InChIKey: LFSXWIRCSGJIBI-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Radioligand Binding Assay: Plasma membranes prepared from HEK 293 EBNA cells transfected with human CB1 or CB2 cannabinoid receptors (3.7 or 3.3 pmol/mg protein, receptor concentration; PerkinElmer) were used for the radioligand binding studies. The composition of incubation buffer for CB1 assay was 50 mM Tris base, 2.5 mM EDTA, 5 mM MgCl2 and 0.5 mg/ml fatty acid free BSA and for CB2 assay was 50 mM Tris base, 2.5 mM EGTA, 5 mM MgCl2 and 1 mg/ml fatty acid free BSA; pH was adjusted to 7.4 by adding 1N HCl. Plasma membranes were diluted with incubation buffer to provide a final protein concentration of 2.4 μg (CB1) or 8 μg (CB2) per well in non-binding surface polystyrene 96-well assay plates (Corning). Compound solutions were prepared in silanized glass tubes and dispensed using pipette tips with SUPERSLIK¿ surface. Competition studies were performed using a final concentration of 0.72 nM [3H]-CP 55,940 (100-180 Ci/mmol, specific activity; PerkinElmer) against test compounds. Non-specific binding was determined using 10 uM of unlabeled CP 55,940. Incubations were performed in 96-well assay plates at a final volume of 200 ul for 90 min (CB1) or 60 min (CB2) at room temperature. Binding reactions were terminated by the addition of ice-cold incubation buffer and rapid filtration through a glass fiber filter using a cell harvester (Inotech). This was followed by 8 additional washes. The glass fiber filter (0.26 mm thickness, 1-1.5 um retention; Inotech) was presoaked in cold incubation buffer containing 0.05% polyethylenimine. The filters were oven-dried at 50° C. for 60 min and counting performed using a Beckman Coulter LS 6500 Multi-Purpose Scintillation Counter (Beckman-Coulter). Binding was determined in duplicate for at least 4 separate experiments.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 441.41, ALogP 3.53, HBA 6, HBD 1, PSA 59.39, QED 0.76
Compound (SMILES): Cc1cc2ccccc2n1-c1nc2c(c(NCc3ccccc3)n1)CNCC2
InChIKey: KEXDPSXGMWVJRT-UHFFFAOYSA-N
Target: Transitional endoplasmic reticulum ATPase (SINGLE PROTEIN), Homo sapiens
UniProt: P55072
Assay: ADP Glo Assay: The ATPase assay is performed according the following protocol: Purified enzyme (20 nM p97), substrate (20 uM ATP) and a dose titration of compounds are mixed in buffer (50 mM TRIS pH 7.5, 20 mM MgCl2, 0.02% TX-100, 1 mM DTT, 0.2% (v/v) glycerol) and incubated at 37 C. for 15 minutes. The reaction is terminated and the level of product generated is measured using the ADP Glo Assay Kit (Promega, Madison Wis.).
Measurement: IC50 < 2000 nM (pChEMBL 5.698999881744385)
Descriptors: MW 369.47, ALogP 3.99, HBA 5, HBD 2, PSA 54.77, QED 0.57
Compound (SMILES): Nc1ccc(-c2ccc(-c3ccccc3S(=O)(=O)N3C[C@@H]4C[C@H]3CO4)cc2F)cn1
InChIKey: NELGVJOKSYDWGT-IRXDYDNUSA-N
Target: Arachidonate 5-lipoxygenase-activating protein (SINGLE PROTEIN), Homo sapiens
UniProt: P20292
Assay: Binding Assay: The assay below is used to test the modulatory activity of compounds against FLAP. Human and mouse FLAP-encoding DNA was amplified by polymerase chain reaction and cloned into pFastBac1 (Invitrogen) with a NH2-terminal 6-His tag for expression in Spodoptera frugiperda (Sf-9) cells. FLAP-containing membranes were prepared as was a FITC-labeled FLAP modulator [5-[({[2-(2-{3-[3-(tert-Butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid]. The FLAP binding assay is performed in HTRF format (homogeneous time resolved fluorescence). FLAP-containing membranes (1 μg/well final for human) are incubated in the presence of the HTRF ligand, [5-[({[2-(2-{3-[3-(tert-butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid] (25 nM final), a terbium labeled anti-His tag antibody (0.5 ng/well final, from Cisbio) and compounds. The reaction is allowed to proceed for two hours after which the plate is read on an Envision plate reader in HTRF mode. Data are expressed as a HTRF ratio.
Measurement: Ki = 32 nM (pChEMBL 7.5)
Descriptors: MW 425.49, ALogP 3.3, HBA 5, HBD 1, PSA 85.52, QED 0.69
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1Cl)N1C2CCCC1c1nnc(-c3ncccn3)n1C2
InChIKey: FCWVNMGJKGGYLS-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Mus musculus
UniProt: Q9Z1M0
Assay: P2X7 FLIPR Assay: 1321N1 cells expressing the recombinant human or rat P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 μl volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250× the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 μL of the compound into 300 μL of assay buffer. A further 3× dilution occurred when transferring 50 μL/well of the compound plate to 100 μL/well in the cell plate. Cells were incubated with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 μL/well of BzATP (final concentration is 250 μM BzATP (human and rat)). The fluorescence change was measured 180 seconds after adding the agonist. Peak fluorescence was plotted as a function of BzATP concentration using Origin 7 software.
Measurement: IC50 = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 448.84, ALogP 4.16, HBA 6, HBD 0, PSA 76.8, QED 0.59
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1Cl)N1[C@H]2CCC[C@@H]1c1nnc(-c3ncccn3)n1C2
InChIKey: FCWVNMGJKGGYLS-SMDDNHRTSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: Ca2+ Flux Assay: 1321N1 cells expressing the recombinant human or rat P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 μl volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250× the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 μL of the compound into 300 μL of assay buffer. A further 3× dilution occurred when transferring 50 μL/well of the compound plate to 100 μL/well in the cell plate. Cells were incubated with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 μL/well of BzATP (final concentration is 250 μM BzATP (human and rat)).
Measurement: Ki = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 448.84, ALogP 4.16, HBA 6, HBD 0, PSA 76.8, QED 0.59
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1Cl)N1[C@@H]2CCC[C@H]1c1nnc(-c3ncccn3)n1C2
InChIKey: FCWVNMGJKGGYLS-RISCZKNCSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: FLIPR Assay: Ca.sup.2+flux: 1321N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 .mu.l volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl.sub.2, 1 MgCl.sub.2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250.times. the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 .mu.L of the compound into 300 .mu.L of assay buffer. A further 3.times. dilution occurred when transferring 50 .mu.L/well of the compound plate to 100 .mu.L/well in the cell plate.
Measurement: IC50 = 8.1 nM (pChEMBL 8.09000015258789)
Descriptors: MW 448.84, ALogP 4.16, HBA 6, HBD 0, PSA 76.8, QED 0.59
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1Cl)N1C2CCCC1c1nnc(-c3ccc(F)cc3)n1C2
InChIKey: NRHCTGLCTSKNEY-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: Ca2+ Flux Assay: 1321N1 cells expressing the recombinant human or rat P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 μl volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250× the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 μL of the compound into 300 μL of assay buffer. A further 3× dilution occurred when transferring 50 μL/well of the compound plate to 100 μL/well in the cell plate. Cells were incubated with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 μL/well of BzATP (final concentration is 250 μM BzATP (human and rat)).
Measurement: Ki = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 464.85, ALogP 5.51, HBA 4, HBD 0, PSA 51.02, QED 0.47
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1Cl)N1[C@H]2CCC[C@@H]1c1nnc(-c3ccc(F)cc3)n1C2
InChIKey: NRHCTGLCTSKNEY-WMLDXEAASA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: Ca2+ Flux Assay: 1321N1 cells expressing the recombinant human or rat P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 μl volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250× the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 μL of the compound into 300 μL of assay buffer. A further 3× dilution occurred when transferring 50 μL/well of the compound plate to 100 μL/well in the cell plate. Cells were incubated with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 μL/well of BzATP (final concentration is 250 μM BzATP (human and rat)).
Measurement: Ki = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 464.85, ALogP 5.51, HBA 4, HBD 0, PSA 51.02, QED 0.47
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1Cl)N1[C@@H]2CCC[C@H]1c1nnc(-c3ccc(F)cc3)n1C2
InChIKey: NRHCTGLCTSKNEY-PBHICJAKSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: FLIPR Assay: Ca.sup.2+flux: 1321N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 .mu.l volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl.sub.2, 1 MgCl.sub.2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250.times. the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 .mu.L of the compound into 300 .mu.L of assay buffer. A further 3.times. dilution occurred when transferring 50 .mu.L/well of the compound plate to 100 .mu.L/well in the cell plate.
Measurement: IC50 = 2322.7 nM (pChEMBL 5.630000114440918)
Descriptors: MW 464.85, ALogP 5.51, HBA 4, HBD 0, PSA 51.02, QED 0.47
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1Cl)N1C2CCCC1c1nnc(-c3cscn3)n1C2
InChIKey: BASXUBNHRSXLCS-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Mus musculus
UniProt: Q9Z1M0
Assay: P2X7 FLIPR Assay: 1321N1 cells expressing the recombinant human or rat P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 μl volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250× the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 μL of the compound into 300 μL of assay buffer. A further 3× dilution occurred when transferring 50 μL/well of the compound plate to 100 μL/well in the cell plate. Cells were incubated with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 μL/well of BzATP (final concentration is 250 μM BzATP (human and rat)). The fluorescence change was measured 180 seconds after adding the agonist. Peak fluorescence was plotted as a function of BzATP concentration using Origin 7 software.
Measurement: IC50 = 15.7 nM (pChEMBL 7.800000190734863)
Descriptors: MW 453.88, ALogP 4.82, HBA 6, HBD 0, PSA 63.91, QED 0.56
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1Cl)N1[C@H]2CCC[C@@H]1c1nnc(-c3cscn3)n1C2
InChIKey: BASXUBNHRSXLCS-IINYFYTJSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Mus musculus
UniProt: Q9Z1M0
Assay: P2X7 Radioligand Binding Assay: human or rat P2X7-1321N1 cells were collected and frozen @ −80° C. On the day of the experiment, cell membrane preparations were made according to standard published methods. The total assay volume was 100 μl:10 μl compound (10×)+(b) 40 μl tracer (2.5×)+50 μl membrane (2×). The tracer used for the assay was tritiated A-804598. The compound can be prepared as described in the literature. (Donnelly-Roberts, D. Neuropharmacology 2008, 56 (1), 223-229.) Compounds, tracer and membranes were incubated for 1 hour @ 4° C. The assay was terminated by filtration (GF/B filters pre-soaked with 0.3% PEI) and washed with washing buffer (Tris-HCl 50 mM). The IC50 generated in the binding assay was corrected for tracer concentration and affinity of the tracer to derive at the affinity (Ki) of the test compounds. The data are presented in Tables 1 and 2 under the headings: P2X7 human Ki (μM) and P2X7 rat Ki (μM). Data are analyzed and graphed on Graphpad Prism 5.
Measurement: Ki = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 453.88, ALogP 4.82, HBA 6, HBD 0, PSA 63.91, QED 0.56
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1Cl)N1[C@@H]2CCC[C@H]1c1nnc(-c3cscn3)n1C2
InChIKey: BASXUBNHRSXLCS-YGRLFVJLSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: Ca2+ Flux Assay: 1321N1 cells expressing the recombinant human or rat P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 μl volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250× the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 μL of the compound into 300 μL of assay buffer. A further 3× dilution occurred when transferring 50 μL/well of the compound plate to 100 μL/well in the cell plate. Cells were incubated with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 μL/well of BzATP (final concentration is 250 μM BzATP (human and rat)).
Measurement: Ki = 398 nM (pChEMBL 6.400000095367432)
Descriptors: MW 453.88, ALogP 4.82, HBA 6, HBD 0, PSA 63.91, QED 0.56
Compound (SMILES): COCC(=O)Nc1cc(COc2ccc(NC(=O)Nc3cc(C(C)(C)C)nn3-c3ccc(C)cc3)c3ccccc23)ccn1
InChIKey: RTDCVLCTBQDLBW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase HCK (SINGLE PROTEIN), Homo sapiens
UniProt: P08631
Assay: Inhibition of HCK (unknown origin) preincubated for 2 hrs followed by FRET peptide addition measured after 1 hr by Z-LYTE assay
Measurement: IC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 592.7, ALogP 6.83, HBA 7, HBD 3, PSA 119.4, QED 0.17
Compound (SMILES): COCC(=O)Nc1cc(Oc2ccc(NC(=O)Nc3cc(C(C)(C)C)nn3-c3ccc(C)cc3)c3ccccc23)ccn1
InChIKey: AVUUHIYJPTWYNX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: c-Src and Syk Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 uL) is incubated with the test compound (either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL, 2.5 uL each) for 2 hr at RT. The FRET peptides (8 uM, 2.5 uL), and appropriate ATP solutions (2.5 uL, 800 uM for c-Src, and 60 uM ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 578.67, ALogP 7.05, HBA 7, HBD 3, PSA 119.4, QED 0.18
Compound (SMILES): COc1ccc(-n2nc(C(C)(C)C)cc2NC(=O)Nc2ccc(Oc3ccnc4[nH]c(=O)cnc34)c3ccccc23)cc1
InChIKey: IBHXVVYFEIGWLJ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase HCK (SINGLE PROTEIN), Homo sapiens
UniProt: P08631
Assay: Fluorescence Resonance Energy Transfer (FRET) Assay: The enzyme inhibitory activity of test compounds was determined by fluorescence resonance energy transfer (FRET) using synthetic peptides labelled with both donor and acceptor fluorophores (Z-LYTE, Invitrogen). Recombinant, phosphorylated p38 MAPK (MAPK12:Millipore) was diluted in HEPES buffer, mixed with compound at the desired final concentrations and incubated for 2 hr at RT. The FRET peptide (2 uM) and ATP (100 uM) were next added to the enzyme/compound mixture and incubated for 1 hr. Development reagent (protease) was added for 1 hr prior to detection in a fluorescence microplate reader. The site-specific protease only cleaves non-phosphorylated peptide and eliminates the FRET signal. Phosphorylation levels of each reaction were calculated using the ratio of coumarin emission (donor) over fluorescein emission (acceptor) with high ratios indicating high phosphorylation levels and low ratios indicating low phosphorylation levels.
Measurement: IC50 = 458 nM (pChEMBL 6.340000152587891)
Descriptors: MW 575.63, ALogP 6.4, HBA 8, HBD 3, PSA 136.05, QED 0.21
Compound (SMILES): CCNC(=O)Nc1ccc2ncc(-c3cc(Cl)c(O)c(OC)c3)nc2n1
InChIKey: CJANMZPZNNYAPL-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Cell-Free Kinase Assay: The inhibitory effect of the compounds according to the invention was tested on various serine/threonine, tyrosine and lipid kinases in enzymatic assays. Recombinant human kinases such as, for example, Erk2, PI3Kalpha, -beta, -gamma, -delta, p38alpha, p38gamma, Jnk1, Jnk2 and others were used in this case, partly as full-length kinases, partly as shortened fragments, but at least consisting of the functional kinase domains. The commercial kinase proteins (Proqinase, Upstate) were used as recombinant fusion proteins with GST (glutathion-S-transferase) or His-Tag. Depending on the type of substrate, the various kinase reactions were quantified by means of suitable ALPHA beads (Perkin-Elmer). The substance testing is described in detail hereinafter for the Erk assay. Selected test results of the Erk2, PD3Kalpha, p38alpha and Jnk2 assays are given below.
Measurement: IC50 = 425 nM (pChEMBL 6.369999885559082)
Descriptors: MW 373.8, ALogP 3.2, HBA 6, HBD 3, PSA 109.26, QED 0.65
Compound (SMILES): CCNC(=O)Nc1ccc2ncc(-c3ccc(O)c(OC)c3)nc2n1
InChIKey: OVGNDEJGXTVQKL-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Cell-Free Kinase Assay: The inhibitory effect of the compounds according to the invention was tested on various serine/threonine, tyrosine and lipid kinases in enzymatic assays. Recombinant human kinases such as, for example, Erk2, PI3Kalpha, -beta, -gamma, -delta, p38alpha, p38gamma, Jnk1, Jnk2 and others were used in this case, partly as full-length kinases, partly as shortened fragments, but at least consisting of the functional kinase domains. The commercial kinase proteins (Proqinase, Upstate) were used as recombinant fusion proteins with GST (glutathion-S-transferase) or His-Tag. Depending on the type of substrate, the various kinase reactions were quantified by means of suitable ALPHA beads (Perkin-Elmer). The substance testing is described in detail hereinafter for the Erk assay. Selected test results of the Erk2, PD3Kalpha, p38alpha and Jnk2 assays are given below.
Measurement: IC50 = 490 nM (pChEMBL 6.309999942779541)
Descriptors: MW 339.36, ALogP 2.55, HBA 6, HBD 3, PSA 109.26, QED 0.67
Compound (SMILES): Cn1cc(CNc2cc(OC[C@H]3C[C@@H]3c3ccccn3)nc3ccnn23)cn1
InChIKey: YLPZTDCVMOWRPZ-CVEARBPZSA-N
Target: Nuclear receptor subfamily 1 group I member 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75469
Assay: Activation of PXR (unknown origin) assessed as CYP3A4 induction
Measurement: EC50 = 23000 nM (pChEMBL 4.639999866485596)
Descriptors: MW 375.44, ALogP 2.65, HBA 8, HBD 1, PSA 82.16, QED 0.53
Compound (SMILES): COc1cc(-c2cc3ncccc3c(O[C@H](C)[C@H]3CNC(=O)C3)n2)ccc1OC(F)(F)F
InChIKey: IWADCUQKACLNKJ-TZMCWYRMSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Kinase Glo Luminescence Kinase Assay: Recombinant human Syk (amino acids 342-635) was expressed as a fusion protein with an N-terminal GST tag, affinity-purified and deep-frozen at a concentration of approx. 50 - 100 uM in test buffer (25 mM HEPES pH7.5; 25 mM MgCI2;5 mM MnCI2; 50 mM KCI; 0.2% BSA; 0.01 % CHAPS; 100 uM Na3V04; 0.5 mM DTT) and 10% glycerol at -80 C until use.The catalytic activity of the GST-Syk kinase fusion protein was determined using the Kinase Glo Luminescence Kinase test (Promega; V6712). In this homogeneous test the amount of ATP remaining after the kinase reaction is quantified by a luciferin-luciferase reaction using luminescence. The luminescence signal obtained correlates with the amount of ATP still present and thus correlates inversely with the activity of the protein kinase. Method:The test compounds were dissolved in 100 % DMSO at a concentration of 10 mM and diluted in DMSO to a concentration of 1 mM.
Measurement: IC50 = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 447.41, ALogP 4.11, HBA 6, HBD 1, PSA 82.57, QED 0.61
Compound (SMILES): Cc1cc2ncccc2c(OC[C@H]2CNC(=O)C2)n1
InChIKey: LGLTUBADVDGOPY-SNVBAGLBSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Kinase Glo Luminescence Kinase Assay: Recombinant human Syk (amino acids 342-635) was expressed as a fusion protein with an N-terminal GST tag, affinity-purified and deep-frozen at a concentration of approx. 50 - 100 uM in test buffer (25 mM HEPES pH7.5; 25 mM MgCI2;5 mM MnCI2; 50 mM KCI; 0.2% BSA; 0.01 % CHAPS; 100 uM Na3V04; 0.5 mM DTT) and 10% glycerol at -80 C until use.The catalytic activity of the GST-Syk kinase fusion protein was determined using the Kinase Glo Luminescence Kinase test (Promega; V6712). In this homogeneous test the amount of ATP remaining after the kinase reaction is quantified by a luciferin-luciferase reaction using luminescence. The luminescence signal obtained correlates with the amount of ATP still present and thus correlates inversely with the activity of the protein kinase. Method:The test compounds were dissolved in 100 % DMSO at a concentration of 10 mM and diluted in DMSO to a concentration of 1 mM.
Measurement: IC50 = 6.2 nM (pChEMBL 8.210000038146973)
Descriptors: MW 257.29, ALogP 1.45, HBA 4, HBD 1, PSA 64.11, QED 0.9
Compound (SMILES): Cc1c([C@@H](O)CN2CCN(C[C@H](O)c3ccc4c(c3C)COC4=O)CC2)ccc2c1COC2=O
InChIKey: OCKGFTQIICXDQW-ZEQRLZLVSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Displacement of [35S]MK-499 from human ERG
Measurement: IC50 = 23000 nM (pChEMBL 4.639999866485596)
Descriptors: MW 466.53, ALogP 2.03, HBA 8, HBD 2, PSA 99.54, QED 0.62
Compound (SMILES): Cc1c([C@H](O)CN2CCN(C[C@@H](O)c3ccc4c(c3C)COC4=O)CC2)ccc2c1COC2=O
InChIKey: OCKGFTQIICXDQW-DNQXCXABSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Inhibition of ROMK (unknown origin) after 30 mins by Tl+ influx assay
Measurement: IC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 466.53, ALogP 2.03, HBA 8, HBD 2, PSA 99.54, QED 0.62
Compound (SMILES): Cc1c([C@H](O)CN2CCN(C[C@H](O)c3ccc4c(c3C)COC4=O)CC2)ccc2c1COC2=O
InChIKey: OCKGFTQIICXDQW-PSWAGMNNSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Inhibition of ROMK (unknown origin) after 30 mins by Tl+ influx assay
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 466.53, ALogP 2.03, HBA 8, HBD 2, PSA 99.54, QED 0.62
Compound (SMILES): O=C(O)c1ccc2c(c1)[nH]c(=O)c1ccncc12
InChIKey: FNBQKJSMQWRTEK-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Chemiluminescent PARP Assay: PARP assays are conducted using a chemiluminescent PARP assay kit (Trevigen). Briefly, reactions are performed in Histone-coated strip wells, by adding 10 microliters test compound dissolved in 1x PARP Buffer (prepared by mixing 20x PARP buffer diluted with high-purity water) and 15 microliters diluted PARP-HSA enzyme (diluted in 1x PARP buffer, 0.1 unit per well) to 25 microliters PARP cocktail (prepared from 10x PARP cocktail and 10x activated DNA, both 2.5 microliters per well and 20 microliters per well of 1x PARP buffer). The reactions are incubated at ambient temperature for 60 minutes, then the liquid was removed. After washing the wells four times with PBS (200 ul), 50 microliters of STREP-HRP (Horseradish Peroxidase) solution (diluted 500-fold in 1x Strep-Diluent) was added and the reactions were allowed to incubate for 30 minutes at ambient temperature.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 240.22, ALogP 1.77, HBA 3, HBD 2, PSA 83.05, QED 0.63
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1Cl)N1C2CCCC1c1nnc(-c3ncccn3)n1C2
InChIKey: FCWVNMGJKGGYLS-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: FLIPR Assay: Ca.sup.2+flux: 1321N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 .mu.l volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl.sub.2, 1 MgCl.sub.2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250.times. the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 .mu.L of the compound into 300 .mu.L of assay buffer. A further 3.times. dilution occurred when transferring 50 .mu.L/well of the compound plate to 100 .mu.L/well in the cell plate.
Measurement: IC50 = 3.8 nM (pChEMBL 8.420000076293945)
Descriptors: MW 448.84, ALogP 4.16, HBA 6, HBD 0, PSA 76.8, QED 0.59
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1Cl)N1[C@H]2CCC[C@@H]1c1nnc(-c3ncccn3)n1C2
InChIKey: FCWVNMGJKGGYLS-SMDDNHRTSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Mus musculus
UniProt: Q9Z1M0
Assay: P2X7 FLIPR Assay: 1321N1 cells expressing the recombinant human or rat P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 μl volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250× the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 μL of the compound into 300 μL of assay buffer. A further 3× dilution occurred when transferring 50 μL/well of the compound plate to 100 μL/well in the cell plate. Cells were incubated with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 μL/well of BzATP (final concentration is 250 μM BzATP (human and rat)). The fluorescence change was measured 180 seconds after adding the agonist. Peak fluorescence was plotted as a function of BzATP concentration using Origin 7 software.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 448.84, ALogP 4.16, HBA 6, HBD 0, PSA 76.8, QED 0.59