lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(Br)cc1[C@@]21N=C(C)C(N)=N1
InChIKey: NYMXIKPEDWDOQL-UYJHFMRCSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: TR-FRET Assay: The β-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human β-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc 10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in −80° C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 μg/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 μM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 μl) was added to the plate then 1 μl of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes. Substrate (10 μl) was then added, mixed and the reaction proceeded for 15 minutes at r.t. The reaction was stopped with the addition of Stop solution (7 μl, NaAcetate, pH 9). The fluorescence of the product was measured on a Victor II plate reader with an excitation wavelength of 340 nm and an emission wavelength of 615 nm. The assay was performed in a Costar 384 well round bottom, low volume, non-binding surface plate (Corning #3676). The final concentration of the enzyme was 2.7 g/ml; the final concentration of substrate was 100 nM (Km of 250 nM). The dimethylsulphoxide control, instead of test compound, defined the 100% activity level and 0% activity was defined by wells lacking enzyme (replaced with reaction buffer). A control inhibitor was also used in dose response assays and had an IC50 of 150 nM.
Measurement: IC50 = 16600 nM (pChEMBL 4.78000020980835)
Descriptors: MW 376.3, ALogP 3.57, HBA 4, HBD 1, PSA 59.97, QED 0.81
Compound (SMILES): COC1CCC2(CC1)Oc1ccc(-c3cccc(Cl)c3)cc1C21N=C(C)C(N)=N1
InChIKey: GYYFFSAIGLHSCV-UHFFFAOYSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: sAPPbeta Release Assay: SH-SY5Y cells were cultured in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids and cryopreserved and stored at −140° C. at a concentration of 7.5-9.5×106 cells per vial. Thaw cells and seed at a conc. of around 10000 cells/well in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids to a 384-well tissue culture treated plate, 100 μL cell susp/well. The cell plates were then incubated for 7-24 h at 37° C., 5% CO2. The cell medium was removed, followed by addition of 30 μL compound diluted in DMEM/F-12 with Glutamax, 10% FCS, 1% non-essential amino acids and 1% PeSt to a final conc. of 1% DMSO. The compounds were incubated with the cells for 17 h (overnight) at 37° C., 5% CO2. Meso Scale Discovery (MSD) plates were used for the detection of sAPPβ release. MSD sAPPβ plates were blocked in 1% BSA in Tris wash buffer (40 μL/well) for 1 h on shake at r.t. and washed 1 time in Tris wash buffer (40 μL/well). 20 μL of medium was transferred to the pre-blocked and washed MSD sAPPβ microplates, and the cell plates were further used in an ATP assay to measure cytotoxicity. The MSD plates were incubated with shaking at r.t. for 2 h and the media discarded. 10 μL detection antibody was added (1 nM) per well followed by incubation with shaking at r.t. for 2 h and then discarded. 40 μL Read Buffer was added per well and the plates were read in a SECTOR Imager.
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 409.92, ALogP 4.71, HBA 5, HBD 1, PSA 69.2, QED 0.78
Compound (SMILES): CC1=NC2(CC(C3CCCOC3)Oc3ccc(Br)cc32)N=C1N
InChIKey: ONKRKKQEVGYQDK-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: TR-FRET Assay: The beta-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human beta-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 ul) was added to the plate then 1 ul of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes. Substrate (10 ul) was then added, mixed.
Measurement: IC50 = 1380 nM (pChEMBL 5.860000133514404)
Descriptors: MW 378.27, ALogP 3.01, HBA 5, HBD 1, PSA 69.2, QED 0.82
Compound (SMILES): CC1=NC2(CC(C3CCCOC3)Oc3ccc(-c4cccc(Cl)c4)cc32)N=C1N
InChIKey: MWVBEWBNSOHPSD-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: TR-FRET Assay: The beta-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human beta-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 ul) was added to the plate then 1 ul of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes. Substrate (10 ul) was then added, mixed.
Measurement: IC50 = 2570 nM (pChEMBL 5.590000152587891)
Descriptors: MW 409.92, ALogP 4.57, HBA 5, HBD 1, PSA 69.2, QED 0.79
Compound (SMILES): CC1=N[C@]2(C[C@H]([C@@H]3CCCOC3)Oc3ccc(-c4cccc(Cl)c4)cc32)N=C1N
InChIKey: MWVBEWBNSOHPSD-ODOSVJCGSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Diluted TR-FRET Assay: TR-FRET AssayThe beta-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human beta-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 ul) was added to the plate then 1 ul of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 409.92, ALogP 4.57, HBA 5, HBD 1, PSA 69.2, QED 0.79
Compound (SMILES): CC1=NC2(CC(C3CCOC(C)(C)C3)Oc3ccc(-c4cccc(Cl)c4)cc32)N=C1N
InChIKey: PNGMSULGCSIWPT-UHFFFAOYSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: sAPPbeta Release Assay: SH-SY5Y cells were cultured in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids and cryopreserved and stored at −140° C. at a concentration of 7.5-9.5×106 cells per vial. Thaw cells and seed at a conc. of around 10000 cells/well in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids to a 384-well tissue culture treated plate, 100 μL cell susp/well. The cell plates were then incubated for 7-24 h at 37° C., 5% CO2. The cell medium was removed, followed by addition of 30 μL compound diluted in DMEM/F-12 with Glutamax, 10% FCS, 1% non-essential amino acids and 1% PeSt to a final conc. of 1% DMSO. The compounds were incubated with the cells for 17 h (overnight) at 37° C., 5% CO2. Meso Scale Discovery (MSD) plates were used for the detection of sAPPβ release. MSD sAPPβ plates were blocked in 1% BSA in Tris wash buffer (40 μL/well) for 1 h on shake at r.t. and washed 1 time in Tris wash buffer (40 μL/well). 20 μL of medium was transferred to the pre-blocked and washed MSD sAPPβ microplates, and the cell plates were further used in an ATP assay to measure cytotoxicity. The MSD plates were incubated with shaking at r.t. for 2 h and the media discarded. 10 μL detection antibody was added (1 nM) per well followed by incubation with shaking at r.t. for 2 h and then discarded. 40 μL Read Buffer was added per well and the plates were read in a SECTOR Imager.
Measurement: IC50 = 597 nM (pChEMBL 6.21999979019165)
Descriptors: MW 437.97, ALogP 5.35, HBA 5, HBD 1, PSA 69.2, QED 0.69
Compound (SMILES): CC#Cc1cncc(-c2ccc3c(c2)C2(N=C(C)C(N)=N2)C2(CCC2)C3)c1
InChIKey: GEFZXXPGNGYLMH-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Diluted TR-FRET Assay: TR-FRET AssayThe beta-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human beta-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 ul) was added to the plate then 1 ul of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 354.46, ALogP 3.83, HBA 4, HBD 1, PSA 63.63, QED 0.79
Compound (SMILES): CCC1=NC2(N=C1N)c1cc(-c3cncc(Cl)c3)ccc1C[C@]21CC[C@@H](OC)CC1
InChIKey: QRKMJBIQBFXXFC-AHLJTMJASA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Diluted TR-FRET Assay: TR-FRET AssayThe beta-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human beta-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 ul) was added to the plate then 1 ul of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes.
Measurement: IC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 422.96, ALogP 4.91, HBA 5, HBD 1, PSA 72.86, QED 0.76
Compound (SMILES): CC#Cc1cncc(-c2ccc3c(c2)C2(N=C(N)C(CC)=N2)[C@]2(CC[C@H](OC)CC2)C3)c1
InChIKey: CGJQMXWLLKPNOI-JZGZJDTKSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: TR-FRET Assay: The β-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human β-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc 10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in −80° C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 μg/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 μM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 μl) was added to the plate then 1 μl of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes. Substrate (10 μl) was then added, mixed and the reaction proceeded for 15 minutes at r.t. The reaction was stopped with the addition of Stop solution (7 μl, NaAcetate, pH 9). The fluorescence of the product was measured on a Victor II plate reader with an excitation wavelength of 340 nm and an emission wavelength of 615 nm. The assay was performed in a Costar 384 well round bottom, low volume, non-binding surface plate (Corning #3676). The final concentration of the enzyme was 2.7 g/ml; the final concentration of substrate was 100 nM (Km of 250 nM). The dimethylsulphoxide control, instead of test compound, defined the 100% activity level and 0% activity was defined by wells lacking enzyme (replaced with reaction buffer). A control inhibitor was also used in dose response assays and had an IC50 of 150 nM.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 426.56, ALogP 4.63, HBA 5, HBD 1, PSA 72.86, QED 0.73
Compound (SMILES): CCC1=NC2(N=C1N)c1cc(-c3cccc(C#N)c3)ccc1C[C@]21CC[C@@H](OC)CC1
InChIKey: LPTYIZIFEMAJPK-AHQGXOPPSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: TR-FRET Assay: The β-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human β-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc 10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in −80° C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 μg/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 μM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 μl) was added to the plate then 1 μl of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes. Substrate (10 μl) was then added, mixed and the reaction proceeded for 15 minutes at r.t. The reaction was stopped with the addition of Stop solution (7 μl, NaAcetate, pH 9). The fluorescence of the product was measured on a Victor II plate reader with an excitation wavelength of 340 nm and an emission wavelength of 615 nm. The assay was performed in a Costar 384 well round bottom, low volume, non-binding surface plate (Corning #3676). The final concentration of the enzyme was 2.7 g/ml; the final concentration of substrate was 100 nM (Km of 250 nM). The dimethylsulphoxide control, instead of test compound, defined the 100% activity level and 0% activity was defined by wells lacking enzyme (replaced with reaction buffer). A control inhibitor was also used in dose response assays and had an IC50 of 150 nM.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 412.54, ALogP 4.73, HBA 5, HBD 1, PSA 83.76, QED 0.79
Compound (SMILES): Cc1nc2ccc(-c3cc(O[C@H](C)[C@H]4CNC(=O)C4)c4cccnc4c3)cc2s1
InChIKey: BIGXOFQDFCWJGU-CXAGYDPISA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: GSK Kinase Assay: Human GSK3beta (expressed and prified from SF21 cells) is obtained from the University Dundee/Scotland (Dr. James Hastie—Dept. of Biochemistry) in 50 mM Tris (pH7.5); 150 mM NaCl; 0.1 mM EGTA, 270 mM Succrose, 0.1% β-mercaptoethanol, 1 mM benzamidine, 0.2 mM PMSF; sequence see below). The enzyme is diluted to 3.56 μM (168 μg/ml) in enzyme dilution buffer and 6 μl aliquots are stored at −80° C.The activity of GSK3 kinase protein is measured using the Z′-LYTETM assay technology from Invitrogen (# PV3324).
Measurement: IC50 = 6820 nM (pChEMBL 5.170000076293945)
Descriptors: MW 403.51, ALogP 4.72, HBA 5, HBD 1, PSA 64.11, QED 0.54
Compound (SMILES): C[C@@H](Oc1cc(-c2ccc3c(c2)ncn3C)cc2ncccc12)[C@H]1CNC(=O)C1
InChIKey: JXJVDYZUMYOTJW-RHSMWYFYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: FLT3 Kinase Assay: FLT3 is obtained from Invitrogen in 50 mM Tris (pH7.5); 100 mM NaCl; 0.05 mM EDTA, 0.05% NP-40, 2 mM DTT; 50% Glycerol # PV3182; Lot 286671; sequence see below). The enzyme is diluted to 720 nM (35 μg/ml) in enzyme dilution buffer and 10 μl aliquots are stored at −80° C.The activity of FLT3 is measured using the Z′-LYTETM assay technology from Invitrogen (#PV3191)
Measurement: IC50 = 267 nM (pChEMBL 6.570000171661377)
Descriptors: MW 386.46, ALogP 3.69, HBA 5, HBD 1, PSA 69.04, QED 0.58
Compound (SMILES): C[C@@H](Oc1cc(-c2ccc3c(c2)CNCC3)cc2ncccc12)[C@H]1CNC(=O)C1
InChIKey: HNASBONLOOCULQ-DNVCBOLYSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: GSK Kinase Assay: Human GSK3beta (expressed and prified from SF21 cells) is obtained from the University Dundee/Scotland (Dr. James Hastie—Dept. of Biochemistry) in 50 mM Tris (pH7.5); 150 mM NaCl; 0.1 mM EGTA, 270 mM Succrose, 0.1% β-mercaptoethanol, 1 mM benzamidine, 0.2 mM PMSF; sequence see below). The enzyme is diluted to 3.56 μM (168 μg/ml) in enzyme dilution buffer and 6 μl aliquots are stored at −80° C.The activity of GSK3 kinase protein is measured using the Z′-LYTETM assay technology from Invitrogen (# PV3324).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 387.48, ALogP 3.45, HBA 4, HBD 2, PSA 63.25, QED 0.72
Compound (SMILES): COc1cc(-c2cc(O[C@H](C)[C@H]3CNC(=O)C3)c3cccnc3c2)ccc1C(F)(F)F
InChIKey: TVBZNTQTCCGAHW-CZUORRHYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: FLT3 Kinase Assay: FLT3 is obtained from Invitrogen in 50 mM Tris (pH7.5); 100 mM NaCl; 0.05 mM EDTA, 0.05% NP-40, 2 mM DTT; 50% Glycerol # PV3182; Lot 286671; sequence see below). The enzyme is diluted to 720 nM (35 μg/ml) in enzyme dilution buffer and 10 μl aliquots are stored at −80° C.The activity of FLT3 is measured using the Z′-LYTETM assay technology from Invitrogen (#PV3191)
Measurement: IC50 = 2270 nM (pChEMBL 5.639999866485596)
Descriptors: MW 430.43, ALogP 4.83, HBA 4, HBD 1, PSA 60.45, QED 0.63
Compound (SMILES): Cc1nc2cc(-c3cc(O[C@H](C)[C@H]4CNC(=O)C4)c4cccnc4c3)ccc2n1C
InChIKey: QBIUCEGPYOMVEE-RDTXWAMCSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: GSK Kinase Assay: Human GSK3beta (expressed and prified from SF21 cells) is obtained from the University Dundee/Scotland (Dr. James Hastie—Dept. of Biochemistry) in 50 mM Tris (pH7.5); 150 mM NaCl; 0.1 mM EGTA, 270 mM Succrose, 0.1% β-mercaptoethanol, 1 mM benzamidine, 0.2 mM PMSF; sequence see below). The enzyme is diluted to 3.56 μM (168 μg/ml) in enzyme dilution buffer and 6 μl aliquots are stored at −80° C.The activity of GSK3 kinase protein is measured using the Z′-LYTETM assay technology from Invitrogen (# PV3324).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 400.48, ALogP 4, HBA 5, HBD 1, PSA 69.04, QED 0.56
Compound (SMILES): Cc1nc(C)c2cc(-c3cc(O[C@H](C)[C@H]4CNC(=O)C4)c4cccnc4c3)ccc2n1
InChIKey: HYXFMJAJQAKJSH-DNVCBOLYSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: GSK Kinase Assay: Human GSK3beta (expressed and prified from SF21 cells) is obtained from the University Dundee/Scotland (Dr. James Hastie—Dept. of Biochemistry) in 50 mM Tris (pH7.5); 150 mM NaCl; 0.1 mM EGTA, 270 mM Succrose, 0.1% β-mercaptoethanol, 1 mM benzamidine, 0.2 mM PMSF; sequence see below). The enzyme is diluted to 3.56 μM (168 μg/ml) in enzyme dilution buffer and 6 μl aliquots are stored at −80° C.The activity of GSK3 kinase protein is measured using the Z′-LYTETM assay technology from Invitrogen (# PV3324).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 412.49, ALogP 4.37, HBA 5, HBD 1, PSA 77, QED 0.54
Compound (SMILES): Cc1nc2cc(-c3cc(O[C@H](C)[C@H]4CNC(=O)C4)c4cccnc4c3)ccc2[nH]1
InChIKey: VBAJQFDWESLMDW-CXAGYDPISA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: GSK Kinase Assay: Human GSK3beta (expressed and prified from SF21 cells) is obtained from the University Dundee/Scotland (Dr. James Hastie—Dept. of Biochemistry) in 50 mM Tris (pH7.5); 150 mM NaCl; 0.1 mM EGTA, 270 mM Succrose, 0.1% β-mercaptoethanol, 1 mM benzamidine, 0.2 mM PMSF; sequence see below). The enzyme is diluted to 3.56 μM (168 μg/ml) in enzyme dilution buffer and 6 μl aliquots are stored at −80° C.The activity of GSK3 kinase protein is measured using the Z′-LYTETM assay technology from Invitrogen (# PV3324).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 386.46, ALogP 3.99, HBA 4, HBD 2, PSA 79.9, QED 0.56
Compound (SMILES): Cc1c(-c2cc(O[C@H](C)[C@H]3CNC(=O)C3)c3cccnc3c2)cnn1C
InChIKey: QUKCRTQLRUGASS-UKRRQHHQSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: GSK Kinase Assay: Human GSK3beta (expressed and prified from SF21 cells) is obtained from the University Dundee/Scotland (Dr. James Hastie—Dept. of Biochemistry) in 50 mM Tris (pH7.5); 150 mM NaCl; 0.1 mM EGTA, 270 mM Succrose, 0.1% β-mercaptoethanol, 1 mM benzamidine, 0.2 mM PMSF; sequence see below). The enzyme is diluted to 3.56 μM (168 μg/ml) in enzyme dilution buffer and 6 μl aliquots are stored at −80° C.The activity of GSK3 kinase protein is measured using the Z′-LYTETM assay technology from Invitrogen (# PV3324).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 350.42, ALogP 2.85, HBA 5, HBD 1, PSA 69.04, QED 0.79
Compound (SMILES): CCCn1cc(-c2cc(O[C@H](C)[C@H]3CNC(=O)C3)c3cccnc3c2)cn1
InChIKey: VRIPIEPFEURRFT-GDBMZVCRSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: GSK Kinase Assay: Human GSK3beta (expressed and prified from SF21 cells) is obtained from the University Dundee/Scotland (Dr. James Hastie—Dept. of Biochemistry) in 50 mM Tris (pH7.5); 150 mM NaCl; 0.1 mM EGTA, 270 mM Succrose, 0.1% β-mercaptoethanol, 1 mM benzamidine, 0.2 mM PMSF; sequence see below). The enzyme is diluted to 3.56 μM (168 μg/ml) in enzyme dilution buffer and 6 μl aliquots are stored at −80° C.The activity of GSK3 kinase protein is measured using the Z′-LYTETM assay technology from Invitrogen (# PV3324).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 364.45, ALogP 3.41, HBA 5, HBD 1, PSA 69.04, QED 0.73
Compound (SMILES): CC(C)n1cc(-c2cc(O[C@H](C)[C@H]3CNC(=O)C3)c3cccnc3c2)cn1
InChIKey: ISOQIZINACKWNJ-GDBMZVCRSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: GSK Kinase Assay: Human GSK3beta (expressed and prified from SF21 cells) is obtained from the University Dundee/Scotland (Dr. James Hastie—Dept. of Biochemistry) in 50 mM Tris (pH7.5); 150 mM NaCl; 0.1 mM EGTA, 270 mM Succrose, 0.1% β-mercaptoethanol, 1 mM benzamidine, 0.2 mM PMSF; sequence see below). The enzyme is diluted to 3.56 μM (168 μg/ml) in enzyme dilution buffer and 6 μl aliquots are stored at −80° C.The activity of GSK3 kinase protein is measured using the Z′-LYTETM assay technology from Invitrogen (# PV3324).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 364.45, ALogP 3.58, HBA 5, HBD 1, PSA 69.04, QED 0.75
Compound (SMILES): CS(=O)(=O)NC(=O)c1cc(F)c(OCC23CC4CC(CC(C4)C2)C3)cc1F
InChIKey: AEFVIYQUYHNRDX-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Radioligand Binding Assay: Radioligand Binding Studies: Saturation experiments. A representative compound of formula (I) was tritiated. Three tritiums were incorporated in place of methyl hydrogens to generate [3H]compound. Binding of this radioligand was preformed in 5 mL borosilicate glass test tubes at room temperature. Binding was initiated by adding membranes to increasing concentrations of [3H]compound in 100 mM NaCl, 20 mM Tris HCl, pH 7.4 buffer containing 0.01% w/v bovine serum albumin (BSA) for 18 h. Non-specific binding was determined in the presence of 1 uM unlabelled compound. After 18 h, the reactants were filtered through GF/C glass fiber filters presoaked in 0.5% w/v polyethylene imine. Filters were washed with 15 mL ice cold 100 mM NaCl, 20 mM Tris HCl, pH7.4 buffer containing 0.25% BSA to separate bound from free ligand. [3H]compound bound to filters was quantified by liquid scintillation counting.Competitive binding experiments.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 399.46, ALogP 3.25, HBA 4, HBD 1, PSA 72.47, QED 0.83
Compound (SMILES): O=C(NO)c1cnc(Nc2cc(F)cc(F)c2)nc1
InChIKey: JPUCLEVGSWEGDV-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: Enzyme Assay: Compounds for testing were diluted in DMSO to 50 fold the final concentration and a ten point three fold dilution series was made. The compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 uM TCEP) to 6 fold their final concentration. The HDAC enzymes (purchased from BPS Biosciences) were diluted to 1.5 fold their final concentration in assay buffer. The tripeptide substrate and trypsin at 0.05 uM final concentration were diluted in assay buffer at 6 fold their final concentration. The final enzyme concentrations used in these assays were 3.3 ng/ml (HDAC1), 0.2 ng/ml (HDAC2), 0.08 ng/ml (HDAC3) and 2 ng/ml (HDAC6). The final substrate concentrations used were 16 uM (HDAC1), 10 uM (HDAC2), 17 uM (HDAC3) and 14 uM (HDAC6). Five ul of compounds and 20 ul of enzyme were added to wells of a black, opaque 384 well plate in duplicate. Enzyme and compound were incubated together at room temperature.
Measurement: IC50 = 177 nM (pChEMBL 6.75)
Descriptors: MW 266.21, ALogP 1.62, HBA 5, HBD 3, PSA 87.14, QED 0.58
Compound (SMILES): CN(c1ncnc2[nH]ccc12)[C@H]1CC[C@H](CS(=O)(=O)N2CCC[C@H](O)C2)CC1
InChIKey: DFVBZOPVGGDURA-JYJNAYRXSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzymatic Assay: A peptide mobility shift assay was used to quantify the phosphorylation of the JAKtide (JAK2 and JAK3) or the IRS-1 peptide (JAK1 and Tyk2). Reactions were carried out in a 384-well plate (Matrical MP-101) in a 10 L total volume. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween-20, ATP (4 M for JAK2 and JAK3, 40 M for JAK1 and 7 M for Tyk2)), 2% DMSO and 1 M peptide substrate (JAKtide for JAK2 and JAK3 and IRS-1 peptide for JAK1 and Tyk2). Compounds were diluted serially in 100% dimethyl sulfoxide and tested in an 11 point dose response in duplicate or quadruplicate (200 nl of compound/DMSO was added per 10 L reaction). The reactions were initiated by the addition of enzyme to the final concentration of 2 nM JAK2, 1 nM JAK3, 12 nM Tyk2 or 20 nM JAK1. The assay was run for 240 minutes for JAK1, 150 minutes for JAK2, 90 minutes for JAK3 and 70 minutes for Tyk2.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 407.54, ALogP 1.74, HBA 6, HBD 2, PSA 102.42, QED 0.78
Compound (SMILES): Cc1cc(C)nc(N2CC3CN(C(=O)c4cccc(F)c4-c4nc(C)no4)CC3C2)n1
InChIKey: ZVMODVWWIRMSHC-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Biological Assay: The in vitro affinity of the compounds for the human orexin-1 and orexin-2 receptors was determined by competitive radioligand binding using [3H]SB SB674042 (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) (Langmead et al., British Journal of Pharmacology 2004, 141:340-346) and [3H]EMPA (N-ethyl-2[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl acetamide) (Malherbe et al., British Journal of Pharmacology, 2009, 156(8), 1326-1341), respectively. The in vitro functional antagonism of the compounds on the human orexin-1 and orexin-2 receptors was determined using fluorometric imaging plate reader (FLIPR) based calcium assays.
Measurement: Ki = 958 nM (pChEMBL 6.019999980926514)
Descriptors: MW 422.46, ALogP 2.8, HBA 7, HBD 0, PSA 88.25, QED 0.64
Compound (SMILES): C[C@@]1(c2cc(NC(=O)c3ccc(Br)cn3)ccc2F)CO[C@@](C)(C(F)(F)F)C(N)=N1
InChIKey: XXUKTXRJOFQHLP-ZWKOTPCHSA-N
Target: Cytochrome P450 2C9 (SINGLE PROTEIN), Homo sapiens
UniProt: P11712
Assay: Inhibition of CYP2C9 in human liver microsomes using diclofenac as substrate incubated 10 mins in presence of NADPH by solid phase extraction mass spectrometry analysis
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 489.27, ALogP 4.16, HBA 5, HBD 2, PSA 89.6, QED 0.63
Compound (SMILES): C[C@@]1(c2cc(NC(=O)c3ncc(C#N)cn3)ccc2F)CO[C@@](C)(C(F)(F)F)C(N)=N1
InChIKey: KSZDQDUDHWUQCJ-ZWKOTPCHSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: Inhibition of wild type human APP751 expressed in CHO cells incubated for 24 hrs by immunoassay
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 436.37, ALogP 2.66, HBA 7, HBD 2, PSA 126.28, QED 0.71
Compound (SMILES): C[C@@]1(c2cc(NC(=O)c3ncc(Br)cn3)ccc2F)CO[C@@](C)(C(F)(F)F)C(N)=N1
InChIKey: MONBOQDZSLNZSN-DLBZAZTESA-N
Target: Cytochrome P450 2C9 (SINGLE PROTEIN), Homo sapiens
UniProt: P11712
Assay: Inhibition of CYP2C9 in human liver microsomes using diclofenac as substrate incubated 10 mins in presence of NADPH by solid phase extraction mass spectrometry analysis
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 490.26, ALogP 3.55, HBA 6, HBD 2, PSA 102.49, QED 0.64
Compound (SMILES): C[C@@]1(c2cc(NC(=O)c3ccc(C#N)cn3)ccc2F)CO[C@@](C)(C(F)(F)F)C(N)=N1
InChIKey: TWKUHRBKUHLJDI-RBUKOAKNSA-N
Target: Cytochrome P450 2D6 (SINGLE PROTEIN), Homo sapiens
UniProt: P10635
Assay: Inhibition of CYP2D6 in human liver microsomes using bufuralol as substrate incubated 10 mins in presence of NADPH by solid phase extraction mass spectrometry analysis
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 435.38, ALogP 3.27, HBA 6, HBD 2, PSA 113.39, QED 0.72
Compound (SMILES): COC1CCC2(CC1)Oc1ccc(-c3cccc(Cl)c3)cc1C21N=C(C)C(N)=N1
InChIKey: GYYFFSAIGLHSCV-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Diluted TR-FRET Assay: TR-FRET AssayThe beta-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human beta-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 ul) was added to the plate then 1 ul of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes.
Measurement: IC50 = 5.3 nM (pChEMBL 8.279999732971191)
Descriptors: MW 409.92, ALogP 4.71, HBA 5, HBD 1, PSA 69.2, QED 0.78
Compound (SMILES): CC1=NC2(CC(C3CCCOC3)Oc3ccc(Br)cc32)N=C1N
InChIKey: ONKRKKQEVGYQDK-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: TR-FRET Assay: The beta-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human beta-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 ul) was added to the plate then 1 ul of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes. Substrate (10 ul) was then added, mixed.
Measurement: IC50 = 8100 nM (pChEMBL 5.090000152587891)
Descriptors: MW 378.27, ALogP 3.01, HBA 5, HBD 1, PSA 69.2, QED 0.82
Compound (SMILES): CC1=NC2(CC(C3CCOC(C)(C)C3)Oc3ccc(-c4cccc(Cl)c4)cc32)N=C1N
InChIKey: PNGMSULGCSIWPT-UHFFFAOYSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: TR-FRET Assay: The β-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human β-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc 10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in −80° C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 μg/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 μM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 μl) was added to the plate then 1 μl of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes. Substrate (10 μl) was then added, mixed and the reaction proceeded for 15 minutes at r.t. The reaction was stopped with the addition of Stop solution (7 μl, NaAcetate, pH 9). The fluorescence of the product was measured on a Victor II plate reader with an excitation wavelength of 340 nm and an emission wavelength of 615 nm. The assay was performed in a Costar 384 well round bottom, low volume, non-binding surface plate (Corning #3676). The final concentration of the enzyme was 2.7 g/ml; the final concentration of substrate was 100 nM (Km of 250 nM). The dimethylsulphoxide control, instead of test compound, defined the 100% activity level and 0% activity was defined by wells lacking enzyme (replaced with reaction buffer). A control inhibitor was also used in dose response assays and had an IC50 of 150 nM.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 437.97, ALogP 5.35, HBA 5, HBD 1, PSA 69.2, QED 0.69
Compound (SMILES): CC#Cc1cncc(-c2ccc3c(c2)C2(N=C(C)C(N)=N2)C2(CCC2)C3)c1
InChIKey: GEFZXXPGNGYLMH-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: TR-FRET Assay: The beta-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human beta-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 ul) was added to the plate then 1 ul of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes. Substrate (10 ul) was then added, mixed.
Measurement: IC50 = 27600 nM (pChEMBL 4.559999942779541)
Descriptors: MW 354.46, ALogP 3.83, HBA 4, HBD 1, PSA 63.63, QED 0.79
Compound (SMILES): CCC1=NC2(N=C1N)c1cc(-c3cncc(Cl)c3)ccc1C[C@]21CC[C@@H](OC)CC1
InChIKey: QRKMJBIQBFXXFC-AHLJTMJASA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: TR-FRET Assay: The beta-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human beta-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 ul) was added to the plate then 1 ul of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes. Substrate (10 ul) was then added, mixed.
Measurement: IC50 = 16300 nM (pChEMBL 4.789999961853027)
Descriptors: MW 422.96, ALogP 4.91, HBA 5, HBD 1, PSA 72.86, QED 0.76
Compound (SMILES): CC#Cc1cncc(-c2ccc3c(c2)C2(N=C(N)C(CC)=N2)[C@]2(CC[C@H](OC)CC2)C3)c1
InChIKey: CGJQMXWLLKPNOI-JZGZJDTKSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: sAPPbeta Release Assay: SH-SY5Y cells were cultured in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids and cryopreserved and stored at −140° C. at a concentration of 7.5-9.5×106 cells per vial. Thaw cells and seed at a conc. of around 10000 cells/well in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids to a 384-well tissue culture treated plate, 100 μL cell susp/well. The cell plates were then incubated for 7-24 h at 37° C., 5% CO2. The cell medium was removed, followed by addition of 30 μL compound diluted in DMEM/F-12 with Glutamax, 10% FCS, 1% non-essential amino acids and 1% PeSt to a final conc. of 1% DMSO. The compounds were incubated with the cells for 17 h (overnight) at 37° C., 5% CO2. Meso Scale Discovery (MSD) plates were used for the detection of sAPPβ release. MSD sAPPβ plates were blocked in 1% BSA in Tris wash buffer (40 μL/well) for 1 h on shake at r.t. and washed 1 time in Tris wash buffer (40 μL/well). 20 μL of medium was transferred to the pre-blocked and washed MSD sAPPβ microplates, and the cell plates were further used in an ATP assay to measure cytotoxicity. The MSD plates were incubated with shaking at r.t. for 2 h and the media discarded. 10 μL detection antibody was added (1 nM) per well followed by incubation with shaking at r.t. for 2 h and then discarded. 40 μL Read Buffer was added per well and the plates were read in a SECTOR Imager.
Measurement: IC50 = 0.81 nM (pChEMBL 9.09000015258789)
Descriptors: MW 426.56, ALogP 4.63, HBA 5, HBD 1, PSA 72.86, QED 0.73
Compound (SMILES): CCC1=NC2(N=C1N)c1cc(-c3cccc(C#N)c3)ccc1C[C@]21CC[C@@H](OC)CC1
InChIKey: LPTYIZIFEMAJPK-AHQGXOPPSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: sAPPbeta Release Assay: SH-SY5Y cells were cultured in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids and cryopreserved and stored at −140° C. at a concentration of 7.5-9.5×106 cells per vial. Thaw cells and seed at a conc. of around 10000 cells/well in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids to a 384-well tissue culture treated plate, 100 μL cell susp/well. The cell plates were then incubated for 7-24 h at 37° C., 5% CO2. The cell medium was removed, followed by addition of 30 μL compound diluted in DMEM/F-12 with Glutamax, 10% FCS, 1% non-essential amino acids and 1% PeSt to a final conc. of 1% DMSO. The compounds were incubated with the cells for 17 h (overnight) at 37° C., 5% CO2. Meso Scale Discovery (MSD) plates were used for the detection of sAPPβ release. MSD sAPPβ plates were blocked in 1% BSA in Tris wash buffer (40 μL/well) for 1 h on shake at r.t. and washed 1 time in Tris wash buffer (40 μL/well). 20 μL of medium was transferred to the pre-blocked and washed MSD sAPPβ microplates, and the cell plates were further used in an ATP assay to measure cytotoxicity. The MSD plates were incubated with shaking at r.t. for 2 h and the media discarded. 10 μL detection antibody was added (1 nM) per well followed by incubation with shaking at r.t. for 2 h and then discarded. 40 μL Read Buffer was added per well and the plates were read in a SECTOR Imager.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 412.54, ALogP 4.73, HBA 5, HBD 1, PSA 83.76, QED 0.79
Compound (SMILES): Cc1nc2ccc(-c3cc(O[C@H](C)[C@H]4CNC(=O)C4)c4cccnc4c3)cc2s1
InChIKey: BIGXOFQDFCWJGU-CXAGYDPISA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: 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 MgCl2; 5 mM MnCl2; 50 mM KCl; either 0.2% BSA or 0.2% HSA; 0.01% CHAPS; 100 uM Na3VO4; 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.MethodThe 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 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 403.51, ALogP 4.72, HBA 5, HBD 1, PSA 64.11, QED 0.54
Compound (SMILES): C[C@@H](Oc1cc(-c2ccc3c(c2)CNCC3)cc2ncccc12)[C@H]1CNC(=O)C1
InChIKey: HNASBONLOOCULQ-DNVCBOLYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: 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 MgCl2; 5 mM MnCl2; 50 mM KCl; either 0.2% BSA or 0.2% HSA; 0.01% CHAPS; 100 uM Na3VO4; 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.MethodThe 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 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 387.48, ALogP 3.45, HBA 4, HBD 2, PSA 63.25, QED 0.72
Compound (SMILES): COc1cc(-c2cc(O[C@H](C)[C@H]3CNC(=O)C3)c3cccnc3c2)ccc1C(F)(F)F
InChIKey: TVBZNTQTCCGAHW-CZUORRHYSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: GSK Kinase Assay: Human GSK3beta (expressed and prified from SF21 cells) is obtained from the University Dundee/Scotland (Dr. James Hastie—Dept. of Biochemistry) in 50 mM Tris (pH7.5); 150 mM NaCl; 0.1 mM EGTA, 270 mM Succrose, 0.1% β-mercaptoethanol, 1 mM benzamidine, 0.2 mM PMSF; sequence see below). The enzyme is diluted to 3.56 μM (168 μg/ml) in enzyme dilution buffer and 6 μl aliquots are stored at −80° C.The activity of GSK3 kinase protein is measured using the Z′-LYTETM assay technology from Invitrogen (# PV3324).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 430.43, ALogP 4.83, HBA 4, HBD 1, PSA 60.45, QED 0.63
Compound (SMILES): Cc1nc2cc(-c3cc(O[C@H](C)[C@H]4CNC(=O)C4)c4cccnc4c3)ccc2n1C
InChIKey: QBIUCEGPYOMVEE-RDTXWAMCSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: 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 MgCl2; 5 mM MnCl2; 50 mM KCl; either 0.2% BSA or 0.2% HSA; 0.01% CHAPS; 100 uM Na3VO4; 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.MethodThe 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 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 400.48, ALogP 4, HBA 5, HBD 1, PSA 69.04, QED 0.56
Compound (SMILES): Cc1nc(C)c2cc(-c3cc(O[C@H](C)[C@H]4CNC(=O)C4)c4cccnc4c3)ccc2n1
InChIKey: HYXFMJAJQAKJSH-DNVCBOLYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: 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 MgCl2; 5 mM MnCl2; 50 mM KCl; either 0.2% BSA or 0.2% HSA; 0.01% CHAPS; 100 uM Na3VO4; 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.MethodThe 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 = 4.9 nM (pChEMBL 8.3100004196167)
Descriptors: MW 412.49, ALogP 4.37, HBA 5, HBD 1, PSA 77, QED 0.54