lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(O)CNC(=O)c1c(=O)oc(O)c2cccc(-c3ccc(C(F)(F)F)cc3)c12
InChIKey: KALGDVSCXIKWNW-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P59722
Assay: Enzyme Assay: HIF-PHD2 activity was measured using homogeneous TR-FRET technology (see also, US2008/004817; Dao J H et al., Anal Biochem. 2009, 384:213-23). To each well of a 1/2 Area 96-well plate was added 2 μL of test compound in DMSO and 40 μL of assay buffer (50 mM Tris PH7.4/0.01% Tween-20/0.1 mg/ml BSA/1 mM Sodium ascorbate/20 μg/ml Catalase/10 μM FeSO4) containing 600 nM full length PHD2. After a 30 min preincubation at room temperature, the enzymatic reactions were initiated by the addition of 8 μL of substrates (final concentrations of 0.2 μM 2-oxoglutarate and 0.5 μM HIF-1α peptide biotinyl-DLDLEMLAPYIPMDDDFQL). After 2 hr at room temperature, the reactions were terminated and signals were developed by the addition of a 50 μL quench/detection mix to a final concentration of 1 mM ortho-phenanthroline, 0.1 mM EDTA, 0.5 nM anti-(His)6LANCE reagent, 100 nM AF647-labeled Streptavidin, and 30 nM (His)6-VHL-elonginB-elonginC complex.
Measurement: IC50 = 90 nM (pChEMBL 7.050000190734863)
Descriptors: MW 407.3, ALogP 3, HBA 5, HBD 3, PSA 116.84, QED 0.61
Compound (SMILES): O=C(O)CNC(=O)c1c(=O)oc(O)c2cccc(-c3cccc(OC(F)(F)F)c3)c12
InChIKey: TYNGBTFQLHIIJC-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P59722
Assay: Enzyme Assay: HIF-PHD2 activity was measured using homogeneous TR-FRET technology (see also, US2008/004817; Dao J H et al., Anal Biochem. 2009, 384:213-23). To each well of a 1/2 Area 96-well plate was added 2 μL of test compound in DMSO and 40 μL of assay buffer (50 mM Tris PH7.4/0.01% Tween-20/0.1 mg/ml BSA/1 mM Sodium ascorbate/20 μg/ml Catalase/10 μM FeSO4) containing 600 nM full length PHD2. After a 30 min preincubation at room temperature, the enzymatic reactions were initiated by the addition of 8 μL of substrates (final concentrations of 0.2 μM 2-oxoglutarate and 0.5 μM HIF-1α peptide biotinyl-DLDLEMLAPYIPMDDDFQL). After 2 hr at room temperature, the reactions were terminated and signals were developed by the addition of a 50 μL quench/detection mix to a final concentration of 1 mM ortho-phenanthroline, 0.1 mM EDTA, 0.5 nM anti-(His)6LANCE reagent, 100 nM AF647-labeled Streptavidin, and 30 nM (His)6-VHL-elonginB-elonginC complex.
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 423.3, ALogP 2.88, HBA 6, HBD 3, PSA 126.07, QED 0.58
Compound (SMILES): O=C(O)CNC(=O)c1c(=O)oc(O)c2cccc(-c3ccc(OC(F)(F)F)cc3)c12
InChIKey: MXZILTYUQNZUTJ-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P59722
Assay: Enzyme Assay: HIF-PHD2 activity was measured using homogeneous TR-FRET technology (see also, US2008/004817; Dao J H et al., Anal Biochem. 2009, 384:213-23). To each well of a 1/2 Area 96-well plate was added 2 μL of test compound in DMSO and 40 μL of assay buffer (50 mM Tris PH7.4/0.01% Tween-20/0.1 mg/ml BSA/1 mM Sodium ascorbate/20 μg/ml Catalase/10 μM FeSO4) containing 600 nM full length PHD2. After a 30 min preincubation at room temperature, the enzymatic reactions were initiated by the addition of 8 μL of substrates (final concentrations of 0.2 μM 2-oxoglutarate and 0.5 μM HIF-1α peptide biotinyl-DLDLEMLAPYIPMDDDFQL). After 2 hr at room temperature, the reactions were terminated and signals were developed by the addition of a 50 μL quench/detection mix to a final concentration of 1 mM ortho-phenanthroline, 0.1 mM EDTA, 0.5 nM anti-(His)6LANCE reagent, 100 nM AF647-labeled Streptavidin, and 30 nM (His)6-VHL-elonginB-elonginC complex.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 423.3, ALogP 2.88, HBA 6, HBD 3, PSA 126.07, QED 0.58
Compound (SMILES): O=C(O)CNC(=O)c1c(=O)oc(O)c2cccc(-c3ccc(Cl)c(Cl)c3)c12
InChIKey: FYICZFUMQLKDAL-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P59722
Assay: Enzyme Assay: HIF-PHD2 activity was measured using homogeneous TR-FRET technology (see also, US2008/004817; Dao J H et al., Anal Biochem. 2009, 384:213-23). To each well of a 1/2 Area 96-well plate was added 2 μL of test compound in DMSO and 40 μL of assay buffer (50 mM Tris PH7.4/0.01% Tween-20/0.1 mg/ml BSA/1 mM Sodium ascorbate/20 μg/ml Catalase/10 μM FeSO4) containing 600 nM full length PHD2. After a 30 min preincubation at room temperature, the enzymatic reactions were initiated by the addition of 8 μL of substrates (final concentrations of 0.2 μM 2-oxoglutarate and 0.5 μM HIF-1α peptide biotinyl-DLDLEMLAPYIPMDDDFQL). After 2 hr at room temperature, the reactions were terminated and signals were developed by the addition of a 50 μL quench/detection mix to a final concentration of 1 mM ortho-phenanthroline, 0.1 mM EDTA, 0.5 nM anti-(His)6LANCE reagent, 100 nM AF647-labeled Streptavidin, and 30 nM (His)6-VHL-elonginB-elonginC complex.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 408.19, ALogP 3.29, HBA 5, HBD 3, PSA 116.84, QED 0.61
Compound (SMILES): O=C(O)CNC(=O)c1c(=O)oc(O)c2cccc(-c3ccc(F)c(F)c3)c12
InChIKey: GJZOTRGBNZONHJ-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P59722
Assay: Enzyme Assay: HIF-PHD2 activity was measured using homogeneous TR-FRET technology (see also, US2008/004817; Dao J H et al., Anal Biochem. 2009, 384:213-23). To each well of a 1/2 Area 96-well plate was added 2 μL of test compound in DMSO and 40 μL of assay buffer (50 mM Tris PH7.4/0.01% Tween-20/0.1 mg/ml BSA/1 mM Sodium ascorbate/20 μg/ml Catalase/10 μM FeSO4) containing 600 nM full length PHD2. After a 30 min preincubation at room temperature, the enzymatic reactions were initiated by the addition of 8 μL of substrates (final concentrations of 0.2 μM 2-oxoglutarate and 0.5 μM HIF-1α peptide biotinyl-DLDLEMLAPYIPMDDDFQL). After 2 hr at room temperature, the reactions were terminated and signals were developed by the addition of a 50 μL quench/detection mix to a final concentration of 1 mM ortho-phenanthroline, 0.1 mM EDTA, 0.5 nM anti-(His)6LANCE reagent, 100 nM AF647-labeled Streptavidin, and 30 nM (His)6-VHL-elonginB-elonginC complex.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 375.28, ALogP 2.26, HBA 5, HBD 3, PSA 116.84, QED 0.64
Compound (SMILES): O=C(O)CNC(=O)c1c(=O)oc(O)c2cccc(-c3cc(F)c(F)c(F)c3)c12
InChIKey: DSAUJJJRDDRPQE-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P59722
Assay: Enzyme Assay: HIF-PHD2 activity was measured using homogeneous TR-FRET technology (see also, US2008/004817; Dao J H et al., Anal Biochem. 2009, 384:213-23). To each well of a 1/2 Area 96-well plate was added 2 μL of test compound in DMSO and 40 μL of assay buffer (50 mM Tris PH7.4/0.01% Tween-20/0.1 mg/ml BSA/1 mM Sodium ascorbate/20 μg/ml Catalase/10 μM FeSO4) containing 600 nM full length PHD2. After a 30 min preincubation at room temperature, the enzymatic reactions were initiated by the addition of 8 μL of substrates (final concentrations of 0.2 μM 2-oxoglutarate and 0.5 μM HIF-1α peptide biotinyl-DLDLEMLAPYIPMDDDFQL). After 2 hr at room temperature, the reactions were terminated and signals were developed by the addition of a 50 μL quench/detection mix to a final concentration of 1 mM ortho-phenanthroline, 0.1 mM EDTA, 0.5 nM anti-(His)6LANCE reagent, 100 nM AF647-labeled Streptavidin, and 30 nM (His)6-VHL-elonginB-elonginC complex.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 393.27, ALogP 2.4, HBA 5, HBD 3, PSA 116.84, QED 0.59
Compound (SMILES): COc1cccc(-c2cccc3c(O)oc(=O)c(C(=O)NCC(=O)O)c23)c1
InChIKey: KUAAWLYMRHJGEW-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P59722
Assay: Enzyme Assay: HIF-PHD2 activity was measured using homogeneous TR-FRET technology (see also, US2008/004817; Dao J H et al., Anal Biochem. 2009, 384:213-23). To each well of a 1/2 Area 96-well plate was added 2 μL of test compound in DMSO and 40 μL of assay buffer (50 mM Tris PH7.4/0.01% Tween-20/0.1 mg/ml BSA/1 mM Sodium ascorbate/20 μg/ml Catalase/10 μM FeSO4) containing 600 nM full length PHD2. After a 30 min preincubation at room temperature, the enzymatic reactions were initiated by the addition of 8 μL of substrates (final concentrations of 0.2 μM 2-oxoglutarate and 0.5 μM HIF-1α peptide biotinyl-DLDLEMLAPYIPMDDDFQL). After 2 hr at room temperature, the reactions were terminated and signals were developed by the addition of a 50 μL quench/detection mix to a final concentration of 1 mM ortho-phenanthroline, 0.1 mM EDTA, 0.5 nM anti-(His)6LANCE reagent, 100 nM AF647-labeled Streptavidin, and 30 nM (His)6-VHL-elonginB-elonginC complex.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 369.33, ALogP 1.99, HBA 6, HBD 3, PSA 126.07, QED 0.63
Compound (SMILES): COc1ccc(-c2cccc3c(O)oc(=O)c(C(=O)NCC(=O)O)c23)cc1
InChIKey: VWJCXGPXZKKYBO-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P59722
Assay: Enzyme Assay: HIF-PHD2 activity was measured using homogeneous TR-FRET technology (see also, US2008/004817; Dao J H et al., Anal Biochem. 2009, 384:213-23). To each well of a 1/2 Area 96-well plate was added 2 μL of test compound in DMSO and 40 μL of assay buffer (50 mM Tris PH7.4/0.01% Tween-20/0.1 mg/ml BSA/1 mM Sodium ascorbate/20 μg/ml Catalase/10 μM FeSO4) containing 600 nM full length PHD2. After a 30 min preincubation at room temperature, the enzymatic reactions were initiated by the addition of 8 μL of substrates (final concentrations of 0.2 μM 2-oxoglutarate and 0.5 μM HIF-1α peptide biotinyl-DLDLEMLAPYIPMDDDFQL). After 2 hr at room temperature, the reactions were terminated and signals were developed by the addition of a 50 μL quench/detection mix to a final concentration of 1 mM ortho-phenanthroline, 0.1 mM EDTA, 0.5 nM anti-(His)6LANCE reagent, 100 nM AF647-labeled Streptavidin, and 30 nM (His)6-VHL-elonginB-elonginC complex.
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 369.33, ALogP 1.99, HBA 6, HBD 3, PSA 126.07, QED 0.63
Compound (SMILES): Cc1cccc(-c2cccc3c(O)oc(=O)c(C(=O)NCC(=O)O)c23)c1
InChIKey: ZAEZVXDATUNLKE-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P59722
Assay: Enzyme Assay: HIF-PHD2 activity was measured using homogeneous TR-FRET technology (see also, US2008/004817; Dao J H et al., Anal Biochem. 2009, 384:213-23). To each well of a 1/2 Area 96-well plate was added 2 μL of test compound in DMSO and 40 μL of assay buffer (50 mM Tris PH7.4/0.01% Tween-20/0.1 mg/ml BSA/1 mM Sodium ascorbate/20 μg/ml Catalase/10 μM FeSO4) containing 600 nM full length PHD2. After a 30 min preincubation at room temperature, the enzymatic reactions were initiated by the addition of 8 μL of substrates (final concentrations of 0.2 μM 2-oxoglutarate and 0.5 μM HIF-1α peptide biotinyl-DLDLEMLAPYIPMDDDFQL). After 2 hr at room temperature, the reactions were terminated and signals were developed by the addition of a 50 μL quench/detection mix to a final concentration of 1 mM ortho-phenanthroline, 0.1 mM EDTA, 0.5 nM anti-(His)6LANCE reagent, 100 nM AF647-labeled Streptavidin, and 30 nM (His)6-VHL-elonginB-elonginC complex.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 353.33, ALogP 2.29, HBA 5, HBD 3, PSA 116.84, QED 0.66
Compound (SMILES): Cc1ccc(-c2cccc3c(O)oc(=O)c(C(=O)NCC(=O)O)c23)cc1
InChIKey: CCVJXBNRIUOSRW-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P59722
Assay: Enzyme Assay: HIF-PHD2 activity was measured using homogeneous TR-FRET technology (see also, US2008/004817; Dao J H et al., Anal Biochem. 2009, 384:213-23). To each well of a 1/2 Area 96-well plate was added 2 μL of test compound in DMSO and 40 μL of assay buffer (50 mM Tris PH7.4/0.01% Tween-20/0.1 mg/ml BSA/1 mM Sodium ascorbate/20 μg/ml Catalase/10 μM FeSO4) containing 600 nM full length PHD2. After a 30 min preincubation at room temperature, the enzymatic reactions were initiated by the addition of 8 μL of substrates (final concentrations of 0.2 μM 2-oxoglutarate and 0.5 μM HIF-1α peptide biotinyl-DLDLEMLAPYIPMDDDFQL). After 2 hr at room temperature, the reactions were terminated and signals were developed by the addition of a 50 μL quench/detection mix to a final concentration of 1 mM ortho-phenanthroline, 0.1 mM EDTA, 0.5 nM anti-(His)6LANCE reagent, 100 nM AF647-labeled Streptavidin, and 30 nM (His)6-VHL-elonginB-elonginC complex.
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 353.33, ALogP 2.29, HBA 5, HBD 3, PSA 116.84, QED 0.66
Compound (SMILES): O=C(O)CNC(=O)c1c(=O)oc(O)c2ccc(-c3cccnc3)cc12
InChIKey: MZIUPCFHSPKVQJ-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P59722
Assay: Enzyme Assay: HIF-PHD2 activity was measured using homogeneous TR-FRET technology (see also, US2008/004817; Dao J H et al., Anal Biochem. 2009, 384:213-23). To each well of a 1/2 Area 96-well plate was added 2 μL of test compound in DMSO and 40 μL of assay buffer (50 mM Tris PH7.4/0.01% Tween-20/0.1 mg/ml BSA/1 mM Sodium ascorbate/20 μg/ml Catalase/10 μM FeSO4) containing 600 nM full length PHD2. After a 30 min preincubation at room temperature, the enzymatic reactions were initiated by the addition of 8 μL of substrates (final concentrations of 0.2 μM 2-oxoglutarate and 0.5 μM HIF-1α peptide biotinyl-DLDLEMLAPYIPMDDDFQL). After 2 hr at room temperature, the reactions were terminated and signals were developed by the addition of a 50 μL quench/detection mix to a final concentration of 1 mM ortho-phenanthroline, 0.1 mM EDTA, 0.5 nM anti-(His)6LANCE reagent, 100 nM AF647-labeled Streptavidin, and 30 nM (His)6-VHL-elonginB-elonginC complex.
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 340.29, ALogP 1.37, HBA 6, HBD 3, PSA 129.73, QED 0.65
Compound (SMILES): O=C(O)CNC(=O)c1c(=O)oc(O)c2ccc(Br)cc12
InChIKey: GFNQOAZDBJEEOM-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P59722
Assay: Enzyme Assay: HIF-PHD2 activity was measured using homogeneous TR-FRET technology (see also, US2008/004817; Dao J H et al., Anal Biochem. 2009, 384:213-23). To each well of a 1/2 Area 96-well plate was added 2 μL of test compound in DMSO and 40 μL of assay buffer (50 mM Tris PH7.4/0.01% Tween-20/0.1 mg/ml BSA/1 mM Sodium ascorbate/20 μg/ml Catalase/10 μM FeSO4) containing 600 nM full length PHD2. After a 30 min preincubation at room temperature, the enzymatic reactions were initiated by the addition of 8 μL of substrates (final concentrations of 0.2 μM 2-oxoglutarate and 0.5 μM HIF-1α peptide biotinyl-DLDLEMLAPYIPMDDDFQL). After 2 hr at room temperature, the reactions were terminated and signals were developed by the addition of a 50 μL quench/detection mix to a final concentration of 1 mM ortho-phenanthroline, 0.1 mM EDTA, 0.5 nM anti-(His)6LANCE reagent, 100 nM AF647-labeled Streptavidin, and 30 nM (His)6-VHL-elonginB-elonginC complex.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 342.1, ALogP 1.08, HBA 5, HBD 3, PSA 116.84, QED 0.77
Compound (SMILES): O=C(O)CNC(=O)c1c(=O)oc(O)c2cccc(Br)c12
InChIKey: CCHAAFYNHWGLRD-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P59722
Assay: Enzyme Assay: HIF-PHD2 activity was measured using homogeneous TR-FRET technology (see also, US2008/004817; Dao J H et al., Anal Biochem. 2009, 384:213-23). To each well of a 1/2 Area 96-well plate was added 2 μL of test compound in DMSO and 40 μL of assay buffer (50 mM Tris PH7.4/0.01% Tween-20/0.1 mg/ml BSA/1 mM Sodium ascorbate/20 μg/ml Catalase/10 μM FeSO4) containing 600 nM full length PHD2. After a 30 min preincubation at room temperature, the enzymatic reactions were initiated by the addition of 8 μL of substrates (final concentrations of 0.2 μM 2-oxoglutarate and 0.5 μM HIF-1α peptide biotinyl-DLDLEMLAPYIPMDDDFQL). After 2 hr at room temperature, the reactions were terminated and signals were developed by the addition of a 50 μL quench/detection mix to a final concentration of 1 mM ortho-phenanthroline, 0.1 mM EDTA, 0.5 nM anti-(His)6LANCE reagent, 100 nM AF647-labeled Streptavidin, and 30 nM (His)6-VHL-elonginB-elonginC complex.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 342.1, ALogP 1.08, HBA 5, HBD 3, PSA 116.84, QED 0.77
Compound (SMILES): Cc1c(N(Cc2ccc(F)c(C(F)(F)F)c2)S(=O)(=O)CCCCBr)sc2ccccc12
InChIKey: IQUHPDZWTJNTCN-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 7.2 nM (pChEMBL 8.140000343322754)
Descriptors: MW 538.43, ALogP 6.88, HBA 3, HBD 0, PSA 37.38, QED 0.18
Compound (SMILES): Cn1ccc2c(-c3cnc(OCC4CCN(C(=O)C5CCNCC5)CC4)c(C(F)(F)F)c3)nc(C#N)nc21
InChIKey: FCWREFNVCZYAOS-UHFFFAOYSA-N
Target: Cathepsin S (SINGLE PROTEIN), Homo sapiens
UniProt: P25774
Assay: Inhibition Assay: 5 μL of a human cathepsin S enzyme (R&D1183-CY-010) was added to a 96-well plate so as to be 20 ng/well. Next, 10-fold dilution of the test compound (a 10 mM DMSO solution) was serially performed with a buffer solution for assay (a solution of 50 mM sodium methoxide, 250 mM sodium chloride and 5 mM dithiothreitol (DTT), adjusted to pH=4.5 with 1 N hydrochloric acid) six times such that the final concentration becomes 0.1 nM to 10 μM then, 20 μL of each of the solutions was added to the wells (the final DMSO concentration was 0.1%), and 25 μL of synthetic substrate VVR-AMC (3211-V manufactured by PEPTIDE INSTITUTE, INC.) was added thereto such that the final concentration becomes 40 μM, thereby starting a enzyme reaction. A fluorescence intensity (an excitation wavelength: 380 nm and a fluorescent wavelength: 460 nm) was measured at 37° C. for 10 minutes immediately after the start of the reaction using a fluorescence spectrophotometer (SPECTRA MAX GEMINI, manu
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 527.55, ALogP 3.54, HBA 8, HBD 1, PSA 108.96, QED 0.54
Compound (SMILES): Cc1ccc(-c2ccccc2)c(C(=O)N2CC[C@H]2c2nnc(-c3ccccc3OC(F)(F)F)[nH]2)c1
InChIKey: CPFLJHAIJBNXIU-NRFANRHFSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Intracellular Calcium Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/mL G418, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM. Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 μL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 μM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement. Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 μL/well, incubated for 120 min and finally 10 μL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist.
Measurement: IC50 = 563 nM (pChEMBL 6.25)
Descriptors: MW 478.47, ALogP 5.93, HBA 4, HBD 1, PSA 71.11, QED 0.39
Compound (SMILES): O=C(Nc1ccc(C2=CCN(C(=O)C3CCOCC3)CC2)cc1)N1Cc2ccncc2C1
InChIKey: DQUFYUFKMPHTTJ-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 5.81 nM (pChEMBL 8.239999771118164)
Descriptors: MW 432.52, ALogP 3.67, HBA 4, HBD 1, PSA 74.77, QED 0.8
Compound (SMILES): Nc1c(/N=N/c2ccc(-c3ccccc3O)nc2)cc(S(=O)(=O)[O-])c2ccccc12.[Na+]
InChIKey: RIMVPJIALPPQMC-QREUMGABSA-M
Target: Transitional endoplasmic reticulum ATPase (SINGLE PROTEIN), Mus musculus
UniProt: Q01853
Assay: Inhibition Assay: Mouse VCP cDNA was added with a DNA sequence corresponding to histidine tag at the amino-terminal, subcloned into a baculovirus vector pVL1392 (BD Bioscience), and expressed in Sf-9 insect cells. The protein was purified with a nickel column (GE Healthcare). The concentration of the protein was adjusted to 0.25-0.5 ug/ml and the protein was stored in a solution containing 50 mM TrisCl pH 8.0, 5 mM EDTA, 10% glycerol, and 15 mM DTT. The ATPase activity was determined as follows. 500 ng of the purified VCP was mixed with 100 uM [gamma-32P]ATP (18.5 GBq/mmol) and the test substance in 20 uL of ATPase buffer (20 mM HEPES (pH7.4), 50 mM KCl, 5 mM MgCl2, 15 mM DTT), and incubated at 37° C. for 10 minutes.The enzyme reaction was stopped with addition of 200 uL of an ice-cold solution containing 7% TCA and 1 mM K2HPO4. 50ul of a solution containing 3.75% ammonium molybdate and 0.02M tungstate silicic/3 NH2SO4 was added, followed by 300 uL of n-butylacetic acid.
Measurement: IC50 = 807 nM (pChEMBL 6.090000152587891)
Descriptors: MW 420.45, ALogP 4.85, HBA 7, HBD 3, PSA 138.23, QED 0.24
Compound (SMILES): O=C(Nc1ccc(N2CCN(C(=O)c3cccnc3)CC2)cc1)N1Cc2ccccc2C1
InChIKey: TVRNANGCIVQYAL-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1251667.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 124 nM (pChEMBL 6.909999847412109)
Descriptors: MW 427.51, ALogP 3.59, HBA 4, HBD 1, PSA 68.78, QED 0.69
Compound (SMILES): CC(=O)N1CCN(c2ccc(CN(C3CCC3)S(=O)(=O)c3cccc(C(C)(C)C)c3)c(F)c2)CC1
InChIKey: WEBRTFHHJOBFGH-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Assay: On day of the assay, 100 uL of 0.05% CHAPS (in deionized H2O) was added to all wells of the GFB Unifilter plate and allowed soak for 1 h. A wash buffer of 50 mM HEPES (pH 7.4), 150 mM NaCl, and 5 mM MgCl2 was prepared to wash the filter plate. To prepare an assay buffer, BSA was added to the wash buffer to reach 0.01% and DTT was added to reach 1 mM. For IC50 mode, 10 mM compound stocks were serially diluted in DMSO with DMSO to give 20x required final concentration in DMSO (15 uL compound+30 uL DMSO). The 20x compound stocks were diluted in DMSO with Assay Buffer 4-fold to reach 5x the final test concentration in 25% DMSO (10 uL compound+30 uL Assay Buffer). Solutions were mixed by aspiration several times with a pipette set on 50 uL volume. For the assay, 10 uL of 5x compound stock solutions in 25% DMSO were added to the assay plate in duplicate.
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 501.67, ALogP 4.54, HBA 4, HBD 0, PSA 60.93, QED 0.58
Compound (SMILES): CC(C)Nc1cc(-n2ncc3cc(C#N)cnc32)ncc1C(=O)NCCC1(O)CC1
InChIKey: RRNFVTMBXIDFAG-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 6.2 nM (pChEMBL 8.210000038146973)
Descriptors: MW 405.46, ALogP 2.15, HBA 8, HBD 3, PSA 128.75, QED 0.55
Compound (SMILES): Cc1cc(-n2cnc3c(NCCC(F)(F)F)cc(Oc4ccccc4)cc32)ccc1C(=O)NC1CC1
InChIKey: XDACBOQYQZWLBB-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Kinase Assay: For the assay 50 nL of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of Mps-1 in assay buffer [0.1 mM sodium-ortho-vanadate, 10 mM MgCl2, 2 mM DTT, 25 mM Hepes pH 7.7, 0.05% BSA, 0.001% Pluronic F-127] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to Mps-1 before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μl of a solution of 16.7 adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μl assay volume is 10 μM) and peptide substrate (1.67 μM=>final conc. in the 5 μl assay volume is 1 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of Mps-1 in the assay was adjusted to the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical enzyme concentrations were in the range of about 1 nM (final conc. in the 5 μl assay volume). The reaction was stopped by the addition of 3 μl of a solution of HTRF detection reagents (100 mM Hepes pH 7.4, 0.1% BSA, 40 mM EDTA, 140 nM Streptavidin-XLent [#61GSTXLB, Fa. Cis Biointernational, Marcoule, France], 1.5 nM anti-phospho(Ser/Thr)-Europium-antibody [#AD0180, Perkin Elmer LAS, Rodgau-Jügesheinn, Germany]. The resulting mixture was incubated 1 h at 22° C. to allow the binding of the phosphorylated peptide to the anti-phospho(Ser/Thr)-Europium-antibody. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Europium-labelled anti-phospho(Ser/Thr) antibody to the Streptavidin-XLent. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured in a Viewlux TR-FRET reader (PerkinElmer LAS, Rodgau-Jügesheinn, Germany).
Measurement: IC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 494.52, ALogP 6.38, HBA 5, HBD 2, PSA 68.18, QED 0.3
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(C#CC3CC3)cc1C21N=C(N)N(CC(=O)OC(C)C)C1=O
InChIKey: IQOVRUKEVDUCMP-MJPADRBUSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Assay: For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham).
Measurement: IC50 = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 463.58, ALogP 2.88, HBA 6, HBD 1, PSA 94.22, QED 0.55
Compound (SMILES): COc1cncc(OC)c1C1CCCC(=O)N1Cc1cccc(-n2cccn2)n1
InChIKey: SRICRIMAFDRKIO-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 3100 nM (pChEMBL 5.510000228881836)
Descriptors: MW 393.45, ALogP 2.93, HBA 7, HBD 0, PSA 82.37, QED 0.64
Compound (SMILES): CN1C(=O)N(c2cccc(C(F)(F)F)c2)C2=C(C(=O)CC2)C1c1ccc(C#N)cc1C(=O)NCc1ncco1
InChIKey: KYYOBXSSHXZTQS-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA.Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 μL of these compound dilutions were mixed with 10 μl Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 μL substrate solution in assay buffer were added (250 μM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths.
Measurement: IC50 = 3.8 nM (pChEMBL 8.420000076293945)
Descriptors: MW 535.48, ALogP 4.73, HBA 6, HBD 1, PSA 119.54, QED 0.5
Compound (SMILES): NC(=O)CC[C@@H]1NC(=O)/C=C\C(=O)NNCCCC(C(N)=O)NCCCCNC(=O)[C@@H](Cc2ccc(C(=O)c3ccccc3)cc2)NC(=O)[C@H](CC2CCCCC2)NC1=O
InChIKey: YZGGXYJEJGRNJU-KNCQIRRISA-N
Target: Insulin-degrading enzyme (SINGLE PROTEIN), Homo sapiens
UniProt: P14735
Assay: IDE Activity Assay: Macrocycles were assayed for IDE inhibition by monitoring cleavage of a substrate peptide containing a fluorophore-quencher pair such that cleavage of the model substrate results in increased fluorescence. Although our selection did not explicitly select for inhibition (only binding is required for enrichment), all four strongly enriched macrocycles inhibited IDE activity with IC50<10uM or equal to 10uM. The most potent macrocycle, trans-A12-B8-C6-D5 (6b), inhibited IDE with an IC50 of 140 nM. The three-dimensional conformation adopted by these compounds appears to be important to their inhibitory activity, as the trans-olefin isomers of macrocycles 3, 5, and 6 exhibit >10-fold stronger potency than the corresponding cis-olefin macrocycle isomers (Table 1). Compounds 1 and 4, which contained glycine (A2) instead of D-benzoylphenylalanine (A12) at the A position were inactive against IDE.
Measurement: IC50 = 5000 nM (pChEMBL 5.300000190734863)
Descriptors: MW 830, ALogP 0.46, HBA 10, HBD 9, PSA 272.81, QED 0.15
Compound (SMILES): O=C(c1ccc2cc(Oc3ccc(C(F)(F)F)cn3)ccc2n1)N1CC(c2cccnc2)C1
InChIKey: PZWDWHPWGNSKMV-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 1040 nM (pChEMBL 5.980000019073486)
Descriptors: MW 450.42, ALogP 5.08, HBA 5, HBD 0, PSA 68.21, QED 0.43
Compound (SMILES): O=C(c1cc(Nc2cc(C3CC3)[nH]n2)nc(NCc2cc(-c3ccccc3)no2)n1)N1CCOCC1
InChIKey: LLINQEQOZSRLOO-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 486.54, ALogP 3.56, HBA 9, HBD 3, PSA 134.09, QED 0.34
Compound (SMILES): CCOCCN1N=C(c2ccc(OC)c(OC)c2)CCCC1=O
InChIKey: MQGMULJGEUKUDZ-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Inhibition of human recombinant PDE4B2 using cAMP as substrate after 2 hrs by TR-FRET assay
Measurement: IC50 = 2270 nM (pChEMBL 5.639999866485596)
Descriptors: MW 320.39, ALogP 2.46, HBA 5, HBD 0, PSA 60.36, QED 0.72
Compound (SMILES): COc1ccc(C2(c3ccc(OC)cc3)NC(=N)N(Cc3ccccc3OC)C2=O)cc1
InChIKey: KPLMJDYWCOGVBO-UHFFFAOYSA-N
Target: Plasmepsin II (SINGLE PROTEIN), Plasmodium falciparum
UniProt: P46925
Assay: Enzyme Inhibition Assay : Plasmepsin-2 (PM-2; Plm II) and Plasmepsin (PM-4; Plm IV) expression and purification was performed following the published protocols (Istvan E S and Goldberg D E, 2005). The final purified protein was activated by diluting the protein to 0.3 mg/mL in activating buffer (0.1 M citrate pH 4.5, 0.1% Tween-20, 50 mM dithiothreitol) and incubated at room temperature for 40 min, then the activated enzyme was diluted in assay buffer (50 mM sodium acetate pH 4.7, 0.01% Tween-20). The enzymatic inhibition reaction was performed in 384 well plates with a total volume of 20 μl. 10 μl of diluted PM-2 or PM-4 enzyme was added to the 384-well plate except blank wells (blank wells add 10 μL of assay buffer) and 20 nL of serials of diluted 1000× compounds were added to the wells with 520 Echo® Liquid Handling System (Labcyte Inc.). 10 μL PM-2 peptide substrate (AnaSpec, Cat#, 62050) with assay buffer was then added to final concentration of 20 μM to start the reaction.
Measurement: IC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 431.49, ALogP 3.52, HBA 5, HBD 2, PSA 83.88, QED 0.6
Compound (SMILES): Cc1nnc(CNC(=O)c2cc(NC(=O)c3cc(-c4ccccn4)c(Cl)cc3Cl)n(-c3ccccc3)n2)o1
InChIKey: HBLQIKLZGCDLAH-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 11.7 nM (pChEMBL 7.929999828338623)
Descriptors: MW 548.39, ALogP 5.11, HBA 8, HBD 2, PSA 127.83, QED 0.29
Compound (SMILES): COc1nc(N)nc2c1ncn2C1OC(COP(=O)(N[C@@H](C)C(=O)OCC(C)C)Oc2ccccc2)[C@@H](O)[C@]12CCO2
InChIKey: LPVCKRYXKXQXSA-FXMYBSOZSA-N
Target: RNA-directed RNA polymerase (SINGLE PROTEIN), Hepacivirus hominis
UniProt: O39930
Assay: HCV Replicon Assay: The HCV NS5B reaction was performed in a 20 μL mixture containing varying concentrations of the test compound, 1 μM of all four natural ribonucleotides, [α-32P]UTP, 20 ng/μL of genotype 1b (−) IRES RNA template, 1 unit/μL of SUPERase·In (Ambion, Austin, Tex.), 40 ng/μL of wild type or S282T NS5B Genotype 1b, 1 mM MgCl2, 0.75 mM MnCl2, and 2 mM DTT in 50 mM Hepes buffer (pH 7.5). The reaction was quenched by adding 80 μL of stop solution (12.5 mM EDTA, 2.25 M NaCl, and 225 mM sodium citrate) after incubating at 27° C. for 30 minutes. The radioactive RNA products were separated from unreacted substrates by passing the quenched reaction mixture through a Hybond N+ membrane (GE Healthcare, Piscataway, N.J.) using a dot-blot apparatus. The RNA products were retained on the membrane and the free nucleotides were washed out. The membrane was washed 4 times with a solution containing 0.6 M NaCl and 60 mM sodium citrate. After rinsing the membrane with water followed by ethanol, the membrane was exposed to a phosphorscreen and the products were visualized and quantified using a phosphorimager.
Measurement: EC50 = 566 nM (pChEMBL 6.25)
Descriptors: MW 606.57, ALogP 2.22, HBA 14, HBD 3, PSA 191.4, QED 0.2
Compound (SMILES): CN1CCC[C@H]1C(=O)N1CCC(CCOc2ccc(-c3nc(C#N)nc4c3ccn4CC3CCOC3)cc2C#N)CC1
InChIKey: GPRRLKYGDFHFAU-JVHFYALYSA-N
Target: Cathepsin S (SINGLE PROTEIN), Homo sapiens
UniProt: P25774
Assay: Inhibition Assay: 5 μL of a human cathepsin S enzyme (R&D1183-CY-010) was added to a 96-well plate so as to be 20 ng/well. Next, 10-fold dilution of the test compound (a 10 mM DMSO solution) was serially performed with a buffer solution for assay (a solution of 50 mM sodium methoxide, 250 mM sodium chloride and 5 mM dithiothreitol (DTT), adjusted to pH=4.5 with 1 N hydrochloric acid) six times such that the final concentration becomes 0.1 nM to 10 μM then, 20 μL of each of the solutions was added to the wells (the final DMSO concentration was 0.1%), and 25 μL of synthetic substrate VVR-AMC (3211-V manufactured by PEPTIDE INSTITUTE, INC.) was added thereto such that the final concentration becomes 40 μM, thereby starting a enzyme reaction. A fluorescence intensity (an excitation wavelength: 380 nm and a fluorescent wavelength: 460 nm) was measured at 37° C. for 10 minutes immediately after the start of the reaction using a fluorescence spectrophotometer (SPECTRA MAX GEMINI, manu
Measurement: IC50 = 7.8 nM (pChEMBL 8.109999656677246)
Descriptors: MW 567.69, ALogP 3.98, HBA 9, HBD 0, PSA 120.3, QED 0.4
Compound (SMILES): Cc1ccccc1CN1Cc2cc3c(cc2C[C@H]1C(=O)N[C@@H](Cc1ccc(-c2ccnc(C)c2C)cc1)C(=O)O)OC[C@H](c1ccc(OCc2ccc(Cl)c(Cl)c2)cc1)O3
InChIKey: CJJTVGOONWAGKQ-IMLAOGSGSA-N
Target: Glucagon-like peptide 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43220
Assay: cAMP Functional Assay: The efficacy of GLP-1 receptor agonists was studied in a cAMP functional assay using HEK-293 Cells expressing the cloned human GLP-1 receptor. GLP-1-expressing cells (10,000-20,000 cells per 0.1 mL) were plated in 96-well plates in Dulbecco's modified eagles media (DMEM) containing 10% fetal bovine serum, 2 mg/mL G418 and penicillin-streptomycin. Following overnight incubation of cells, media was removed, and compounds (at concentrations ranging from 0.0001 to 100 μM) were added to monolayer cells in Iscove's modified dulbecco's medium (IMDM), 100 μM RO 20-1724 PDE inhibitor, 0.1% BSA, 2% DMSO in a final volume of 100 μL, and incubated for 30 min at 37° C. 95%02, 5% CO2 in a humidified incubator. Alternatively, cells were harvested and combined with compounds in suspension using the buffer and protocol described above. cAMP was quantified using a homogenous time-resolved fluorescence detection system (cAMP dynamic, CIS bio International).
Measurement: EC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 842.82, ALogP 9.81, HBA 7, HBD 2, PSA 110.22, QED 0.13
Compound (SMILES): CCN(CC)C1CCN(c2ccc(Nc3ncc4cc5n(c4n3)C3(CCCCC3)CNC5=O)nc2)CC1
InChIKey: LWCQVHGNSZVWQE-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P24941
Assay: Caliper Assay: Selected compounds disclosed herein were tested in CDK4/cyclinD1, CDK6/CycD3 CDK2/CycA and CDK2/cyclinE kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Phosphoacceptor substrate peptide concentration used was 1 μM for all assays and Staurosporine was used as the reference compound for all assays. Specifics of each assay are as described below:CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM; Incubation time: 3 hr.CDK2/CyclinE: Enzyme concentration: 0.28 nM; ATP concentration: 100 μM; Incubation time: 1 hr.CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 10 hr.CDK6/CyclinD3: Enzyme concentration: 1 nM; ATP concentration: 300 μM; Incubation time: 3 hr.
Measurement: IC50 = 1250 nM (pChEMBL 5.900000095367432)
Descriptors: MW 502.67, ALogP 4.28, HBA 8, HBD 2, PSA 91.21, QED 0.52
Compound (SMILES): CCCN(Cc1ccc(OCCN2C(=O)CCC2=O)c(C)c1)C(CC(=O)O)c1ccc(Cl)cc1
InChIKey: QFFORRJHQQNWMP-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Displacement of [125I]-CXCL10 from human recombinant CXCR3 transfected in Flp-In-CHO cell membranes after 60 mins by gamma counting method
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 487, ALogP 4.6, HBA 5, HBD 1, PSA 87.15, QED 0.44