lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cc(N2C(=O)NC(=O)C23COC3)cc(C)c1CCS(=O)(=O)N1CCC2(CC1)N=C(c1ccc(OC(F)(F)F)cc1)NC2=O
InChIKey: WMHJWHBMOVKVON-UHFFFAOYSA-N
Target: Parathyroid hormone/parathyroid hormone-related peptide receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: P25961
Assay: cAMP Assay: Instead of HKRK-B7 cells, LLC-PK46_RATO_PTH1R cells overexpressing rat PTH1R, which were established at Chugai Pharmaceutical, were used to take measurements in a similar manner to Test Example 1: HKRK-B7 or LLC-PK1 cells were seeded into a 96-well plate at 1×105 cells/well and incubated overnight. On the following day, 50 μl of cAMP assay buffer (DMEM, 2 mM IBMX, 0.2 mg/ml bovine serum albumin, 35 mM Hepes-NaOH, pH 7.4) containing human PTH(1-34) or each compound was added and the plate was placed in a 37° C. incubator. The cells were incubated for 20 minutes. After removing the medium, the cells were washed with 100 μl of cAMP assay buffer once. The plate was placed on dry ice powder to freeze the cells and then removed from the dry ice. The cells were lysed with 40 μl of 50 mM HCl and frozen again on dry ice. The amount of intracellular cAMP produced was measured using a commercially available cAMP EIA kit (Biotrack cAMP EIA system, GE health care).
Measurement: EC50 = 12000 nM (pChEMBL 4.920000076293945)
Descriptors: MW 649.65, ALogP 2.31, HBA 8, HBD 2, PSA 146.71, QED 0.44
Compound (SMILES): COC(=O)N[C@@H](C)CNc1nccc(C2=C(c3cccc(NS(=O)(=O)c4c(F)cccc4F)c3F)C(=O)C(C)(C)O2)n1
InChIKey: UMYDEXXWHULLMG-AWEZNQCLSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: LanthaScreen Kinase Assay: The BRAFV600E enzymatic assay was performed using a LanthaScreen kinase assay kit purchased from Life Technologies (Grand Island, N.Y.). The assay was conducted according to the procedure provided in the assay kit. In brief, the enzyme reaction was carried out in the kinase reaction buffer containing BRAFV600E (20 ng/mL), ATP (2 uM), Fluorescein-MAP2K1 inactive substrate (0.4 uM), HEPES (50 mM, pH 7.5), 0.01% BRIJ-35, MgCl2 (10 mM), and EGTA (1 mM) in the presence or absence of the tested compounds at various concentrations in a 384-well plate at room temperature (22±1° C.) for 60 minutes. The final reaction volume for each reaction was 10 ul. The reaction was stopped by addition of 10 ul of TR-FRET dilution buffer kinase supplemented with kinase quench buffer (10 mM final) and Tb-anti-pMAP2K1 (2 nM final). The plate was further incubated at room temperature for another 60 minutes, and the fluorescent signals were read on Victor 5 (Perkin Elmer) with excitation at 340 nM and emission at 495 and 520 nM. The assay signal was determined as a ratio of FRET-specific signal measured with emission filter at 520 nM to that of the signal measured with Tb-specific emission filter at 495 nM. IC50 value was calculated using appropriate programs in GraphPad Prism by plotting the logarithm of the concentration versus percent inhibition.
Measurement: IC50 = 97 nM (pChEMBL 7.010000228881836)
Descriptors: MW 605.6, ALogP 4.1, HBA 9, HBD 3, PSA 148.61, QED 0.33
Compound (SMILES): O=C(c1n[nH]c2c1CCN(CC1CC1)C2)N1CCC(c2cccc(F)c2C(F)(F)F)CC1
InChIKey: WYOYZAUCDDVZCO-UHFFFAOYSA-N
Target: Retinol-binding protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P02753
Assay: Scintillation Proximity Binding Assay: Untagged human RBP4 purified from urine of tubular proteinuria patients was purchased from Fitzgerald Industries International. It was biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation kit from Pierce following the manufacturer's recommendations. Binding experiments were performed in 96-well plates (OptiPlate, PerkinElmer) in a final assay volume of 100 ul per well in SPA buffer (IX PBS, pH 7.4, 1 mM EDTA, 0.1% BSA, 0.5% CHAPS). The reaction mix contained 10 nM 3H-Retinol (48.7 Ci/mmol; PerkinElmer), 0.3 mg/well Streptavidin-PVT beads, 50 nM biotinylated RBP4 and a test compound. Nonspecific binding was determined in the presence of 20 uM of unlabeled retinol. The reaction mix was assembled in the dark under dim red light. The plates were sealed with clear tape (TopSeal-A: 96-well microplate, PerkinElmer), wrapped in the aluminum foil, and allowed to equilibrate 6 hours at room temperature followed by overnight incubation at +4° C. Radiocounts were measured using a TopCount NXT counter (Packard Instrument Company).
Measurement: IC50 = 4.345 nM (pChEMBL 8.359999656677246)
Descriptors: MW 450.48, ALogP 4.36, HBA 3, HBD 1, PSA 52.23, QED 0.7
Compound (SMILES): Cc1cc2c(c3ncccc13)NS(=O)(=O)c1ccccc1-2
InChIKey: URKNCOJGQCVDGA-UHFFFAOYSA-N
Target: Ferroportin (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NP59
Assay: Antagonist activity at C-terminal halo-tag fused human ferroportin expressed in MDCK cells assessed as inhibition of hepcidin-induced ferroportin internalization after overnight incubation by Draq5 staining-based fluorescence assay
Measurement: IC50 = 599 nM (pChEMBL 6.21999979019165)
Descriptors: MW 296.35, ALogP 3.32, HBA 3, HBD 1, PSA 59.06, QED 0.69
Compound (SMILES): COc1cc(CN(C[C@H]2CC[C@H](C(=O)O)CC2)C(C)c2ccc3c(c2)CCO3)ccc1OCCN1C(=O)CCC1=O
InChIKey: WDNRFEUNXWKOAC-WDDOAMHJSA-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 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 564.68, ALogP 4.61, HBA 7, HBD 1, PSA 105.61, QED 0.37
Compound (SMILES): COCC(C)(C)Nc1nc2c(-c3cc4c([nH]3)CCNC4=O)cccc2nc1C
InChIKey: MDZQPPYWTULKAT-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 384-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 4.3 nM (pChEMBL 8.369999885559082)
Descriptors: MW 379.46, ALogP 3.06, HBA 5, HBD 3, PSA 91.93, QED 0.63
Compound (SMILES): COCc1nc2c(c(Nc3ccc(C(F)(F)F)nc3)n1)CCN(c1ncccc1C(F)(F)F)CC2
InChIKey: JZMZGTGLHAKUMG-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Functional Assay: HEK293 cells were transfected with human TRPV1 cloned in pcDNA3.1zeo(+) using the Effectene non-liposomal lipid based transfection kit (Qiagen) (hTRPV1/HEK293). hTRPV1/HEK293 cells were routinely grown as monolayers under selection in zeocin (200 μg/mL; Invitrogen) in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, and penicillin/streptomycin (50 units/mL) in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 1380 nM (pChEMBL 5.860000133514404)
Descriptors: MW 498.43, ALogP 4.8, HBA 7, HBD 1, PSA 76.06, QED 0.51
Compound (SMILES): CC(C)(C)c1cccc(Nc2ccccc2-c2nnn[nH]2)c1
InChIKey: UVQQSBCPOHKBSX-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 18 (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z418
Assay: Activation of human TRESK channel expressed in HEK293 cells assessed as induction of channel current by whole cell patch clamp assay
Measurement: EC50 = 18800 nM (pChEMBL 4.730000019073486)
Descriptors: MW 293.37, ALogP 3.91, HBA 4, HBD 2, PSA 66.49, QED 0.77
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CC[C@H]2c2noc(-c3cccc(F)c3C)n2)c1
InChIKey: XODWTMJEFQLUSV-IBGZPJMESA-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 = 655 nM (pChEMBL 6.179999828338623)
Descriptors: MW 418.43, ALogP 3.66, HBA 7, HBD 0, PSA 89.94, QED 0.5
Compound (SMILES): Cc1ccc(C(=O)N2C[C@@H](C)OC[C@H]2Cc2cc(-c3ncccn3)ccc2F)c(-n2nccn2)c1C
InChIKey: SWDMQWHTMPUUHA-XMSQKQJNSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: OX Inhibition 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 ug/mL G418, 100 U/mL penicillin, 100 ug/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.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 486.55, ALogP 3.95, HBA 7, HBD 0, PSA 86.03, QED 0.42
Compound (SMILES): FC(F)(F)c1ccc(Nc2nc(N3CCN(Cc4ccccc4)CC3)nc3c2CCN(c2ncccc2C(F)(F)F)CC3)cc1
InChIKey: HYZPJGLHFHZDQO-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Functional Assay: HEK293 cells were transfected with human TRPV1 cloned in pcDNA3.1zeo(+) using the Effectene non-liposomal lipid based transfection kit (Qiagen) (hTRPV1/HEK293). hTRPV1/HEK293 cells were routinely grown as monolayers under selection in zeocin (200 μg/mL; Invitrogen) in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, and penicillin/streptomycin (50 units/mL) in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 627.64, ALogP 6.58, HBA 7, HBD 1, PSA 60.42, QED 0.24
Compound (SMILES): CCC1CC(=O)NN=C1c1ccc2nc(-c3ccc(OCC4CCCCC4)cc3)oc2c1
InChIKey: PYNSEOVAOBZFNF-UHFFFAOYSA-N
Target: Glucose-dependent insulinotropic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDV5
Assay: AlphaScreen cAMP Assay: MIN6 cells [Miyazaki J et al. Endocrinology. 1990 July; 127(1):126-32] were stably transfected with an expression vector for human GPR119 cDNA (Acc. No. NP-848566). Min-6/hGPR119 cells are cultured in DMEM, 10% FBS, 50 uM beta-mercaptoethanol, 0.3 mg/mL Geniticin, 2 mM GlutaMAX at 37 C. 5% CO2. For the assay, the cells are placed in Optiplates (white, 384-well, 160W-barcoded, TC, sterile with lid, Cat. No. #6007688 (Perkin Elmer); 10000 cells/well; 50 ul). The plates covered with lids are then incubated for 24 hours at 37 C./5% CO2.After the medium has been sucked out of the wells completely, 10 ul of the test compound are added, the compounds are diluted beforehand with stimulating buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 5 mM NaHCO3; pH 7.4. 0.5 mM IBMX and 0.1% BSA, the final DMSO concentration is 1%). After 45 minutes' incubation at RT, the cAMP concentrations are determined using the AlphaScreen cAMP Assay.
Measurement: EC50 = 145 nM (pChEMBL 6.840000152587891)
Descriptors: MW 431.54, ALogP 5.7, HBA 5, HBD 1, PSA 76.72, QED 0.54
Compound (SMILES): Cc1cc(CCc2cc(O)c(=O)[nH]n2)ccc1C(F)(F)F
InChIKey: YGZOVSTVOIYPPU-UHFFFAOYSA-N
Target: D-amino-acid oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P14920
Assay: In Vitro DAAO Enzyme Assay: Human DAAO enzyme was supplied by the Takeda Pharmaceutical Company (Osaka) and each batch was tested and used at concentrations giving comparable levels of activity. The Km of D-Serine was measured for each enzyme batch to maintain consistency; this Km was used in subsequent assays.On the day of the assay compounds were serially diluted in DMSO before being diluted 1:20 with assay buffer (20 mM Tris ph 7.4). A 5 ul portion of assay buffer was added to the wells of a 384 clear base black-walled plate (Corning), 5 ul of diluted compound was then added via automated plate to plate transfer using the Bravo liquid handler (Agilent technologies) followed by 5 ul of human DAAO enzyme and then 5 ul D-Serine 50 mM was added to all but the negative control wells (final concentration of 10 mM). Finally 5 ul Amplex red reagent (Invitrogen) was added to all wells as per manufacturer's protocol. The plate was incubated for 60 minutes in the dark at 25° C., and the fluorescence in each well was measured in the Envision plate reader.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 298.26, ALogP 2.59, HBA 3, HBD 2, PSA 65.98, QED 0.92
Compound (SMILES): CC1CCN(c2cncc(-c3c[nH]c4ccc(-c5nnc(N)s5)cc34)n2)CC1
InChIKey: IIFMWBFXGMFSOH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 12.72 nM (pChEMBL 7.900000095367432)
Descriptors: MW 391.5, ALogP 3.96, HBA 7, HBD 2, PSA 96.61, QED 0.55
Compound (SMILES): O=C(CN[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1)NCC(F)(F)F
InChIKey: XNSIBQZZWITRMV-XYPYZODXSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 395.39, ALogP 1.9, HBA 6, HBD 3, PSA 100, QED 0.61
Compound (SMILES): Nc1nnc(-c2ccc3[nH]cc(-c4nccc(-c5ccccc5)n4)c3c2)o1
InChIKey: UYWOVNUCOZQXAD-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 6.846 nM (pChEMBL 8.15999984741211)
Descriptors: MW 354.37, ALogP 3.92, HBA 6, HBD 2, PSA 106.51, QED 0.51
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3sc4cc(F)ccc4c3Cl)CC2)C1
InChIKey: GPKMLSFODGOOPI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Inhibition of human N-terminal His-tagged JAK1 catalytic domain (837 to 1142 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 <= 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 575.07, ALogP 5.06, HBA 8, HBD 1, PSA 106.73, QED 0.31
Compound (SMILES): Nc1ncnc(N2CCC(c3nc(-c4ccnc(C(F)(F)F)c4)cn3CCN3CCC3)CC2)c1OCC(F)(F)F
InChIKey: CUNFBMBOBMNCLK-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 9.7 nM (pChEMBL 8.010000228881836)
Descriptors: MW 570.54, ALogP 4.37, HBA 9, HBD 1, PSA 98.22, QED 0.4
Compound (SMILES): C[C@@H](NC(=O)c1ccc2c(=O)n(-c3ccc(F)cc3)c(CCCCC(=O)O)cc2c1)c1ccc(F)cc1
InChIKey: NXJVSPCZMLVTEC-GOSISDBHSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Binding Assay: Radioligand binding was performed using filtration or scintillation proximity assay (SPA) technology. SPA assays were done at room temperature in 10 mM HEPES pH 7.4, 1 mM EDTA containing 2 mM MnCl2 and 16 nM [3H]-PGD2 (PerkinElmer, Waltham, Mass.) (164 Ci mmol-1), in a final volume of 0.05 mL. Competing ligands were diluted in dimethylsulfoxide and added using very low volumes (500 nL). The reaction was initiated by the addition of a mixture of 3.52 ug of membrane protein prepared from a human embryonic kidney (HEK)-hCRTH2 cell line adhered to 140 ug of wheatgerm agglutinin SPA beads (PerkinElmer). Total and non-specific binding were determined in the absence and the presence of a CRTH2 antagonist, respectively. The reaction was routinely conducted for 60 minutes at room temperature followed by centrifugation for 5 minutes at 1000 RPM. The radioactivity was measured with a TopCountNXT (PerkinElmer). Filtration binding assays were done in a similar way with minor differences. The assay volume was 0.20 mL and competing ligands in dimethylsulfoxide were added in 2 uL. [3H]-PGD2 was used at 0.6 nM, and reactions were initiated by the addition of 10 ug of membrane protein. Reactions were terminated by filtration through GF/C filter plates (PerkinElmer) presoaked in 10 mM HEPES. After washing with buffer, plates were dried in a 50° C. oven for 1 h, scintillation cocktail was added and radioactivity was measured. Results determined using the SPA-based assay were similar to those from the filtration binding assay.
Measurement: Ki = 5.159 nM (pChEMBL 8.289999961853027)
Descriptors: MW 504.53, ALogP 5.56, HBA 4, HBD 2, PSA 88.4, QED 0.29
Compound (SMILES): NCCNc1cc(-c2n[nH]c3cnc(-c4cccnc4)cc23)ccn1
InChIKey: QQEGBGHTXJUJDN-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.
Measurement: Ki = 0.941 nM (pChEMBL 9.029999732971191)
Descriptors: MW 331.38, ALogP 2.45, HBA 6, HBD 3, PSA 105.4, QED 0.52
Compound (SMILES): CC1(C)COCCN1C(=O)c1ccc(N2CC(F)(F)C2)c(OCC2CC2)n1
InChIKey: HKYMBPFAXUPGAT-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37 °C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30 °C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30 °C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 5.2 nM (pChEMBL 8.279999732971191)
Descriptors: MW 381.42, ALogP 2.58, HBA 5, HBD 0, PSA 54.9, QED 0.78
Compound (SMILES): CC(=O)N(C)CCCn1c(-c2ccccc2)cc(C(=O)Nc2cccc(C(F)(F)F)c2)c1C
InChIKey: GLQOHZMCKNLAGM-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Inhibition Assay: Test Example 1: (1) The TRPV4 inhibitory activity was measured using CHO-K1 cells stably expressing human TRPV4 (hTRPV4/CHO cells). (2) In the day before the experimental day, frozen cell were thawed and washed with the culture medium (MEM-α, 10% FBS, 2 mmol/L GlutaMax, 50 unit Penicillin, 50 μg/mL Streptomycin). Then cells were suspended in the culture medium. (3) The 384-well plates which hTRPV4/CHO cells were seeded at densities of 4000 cells/well in the culture medium and cultured in a CO2 incubator in the presence of 5% CO2 at 37° C. for overnight was used as assay plates. (4) The assay plate was washed with assay buffer (Hanks, 20 mmol/L HEPES, 2.5 mmol/L probenecid, pH7.4), and the buffer were remained at 20 μL/well. (5) 10 μl of dye loading buffer (9 μmol/L Fluo 4-AM, 0.09% Pluronic F-127/assay buffer) was added to each well, then the assay plate was incubated in a CO2 incubator in the presence of 5% CO2 at 37° C. for one hour. (final 3 μmol/L Fluo4-AM). (6) The assay plate was washed with the assay buffer, and the buffers were remained at 20 μL/well. Then the assay plate was incubated for 10 min at room temperature. (7) Diluted compound solution was dispensed to each well in the compound plate, and mixed with built-in Pipette and Mixer of the Fluorescence analysis system FLIPR TETRA (Molecular devices). (8) After incubation for 5 min, 20 μL of 4α-PDD solution were applied to each well of assay plate and mixed with the FLIPR TETRA. (final 1 Î¼mol/L 4α-PDD). (9) The fluorescent intensity was measured with FLIPR TETRA system for 10 min from the point of time addition the compound solution, at Ex 470-495 nm, Em 515-575 nm wavelength.
Measurement: IC50 = 590 nM (pChEMBL 6.230000019073486)
Descriptors: MW 457.5, ALogP 5.6, HBA 3, HBD 1, PSA 54.34, QED 0.5
Compound (SMILES): COc1ccccc1CN1CCC(CCN2C(=O)c3cc4c(cc3C23CC3)OCO4)CC1
InChIKey: XZYXZCLESLLRHB-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Mus musculus
UniProt: P21836
Assay: In Vitro Pharmacodynamic Assay: Modified Ellman method (Alvin V. et al. JWS-USC-75-IX Improves Information Processing and Cognitive Function in Animal Models[J]. Journal of Pharmacology and experimental therapeutics, 2010, 336(3): 751) was employed in the test, for testing the inhibition effect of a series of compounds of formula (I) on acetylcholinesterase, so that active AchEIs were screened out, and their activity were evaluated via the IC50 value of inhibition of the series of compounds of formula (I) on the enzyme.
Measurement: IC50 > 2000 nM (pChEMBL 5.698999881744385)
Descriptors: MW 434.54, ALogP 4.17, HBA 5, HBD 0, PSA 51.24, QED 0.68
Compound (SMILES): CC(c1ccsc1)c1ccc(O)cc1O
InChIKey: KTLHIVVOBPWRRH-UHFFFAOYSA-N
Target: Polyphenol oxidase 1 (SINGLE PROTEIN), Agaricus bisporus
UniProt: Q00024
Assay: Inhibition Assay: Reader: Synergy HT program: tyrosinase 280-490 kinetics: kinetics over 45 minutes, reading at t=10 minutes, Tests in transparent 96-well plates, Phosphate buffer (pH 6.8), Enzyme: mushroom tyrosinase (T-3824, Sigma), Substrate: L-tyrosine (T-3754, Sigma), Positive control: Kojic acid (KA) (60890, Fluka) (reference inhibitor). Reference Molecules for the Test: Kojic acid: 9 μM<IC50<20 μM (PPO), 3 &muM<IC50<7 μM (MPMO); Vitamin C: 20 μM<IC50<40 μM (PPO); Reduced glutathione: 55% inhibition at 25 μM (PPO), IC50=1-2 μM (MPMO); Hydroquinone: IC50=3-4 μM (MPMO); Arbutin: 57% inhibition at 88 μM (MPMO).
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 220.29, ALogP 3.31, HBA 3, HBD 2, PSA 40.46, QED 0.81
Compound (SMILES): NCCCNC(=O)c1cc(NC(=O)c2cc(-c3ncccc3F)ccc2Cl)n(-c2ccccc2)n1
InChIKey: LWELCNSHHUYGET-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 = 85.5 nM (pChEMBL 7.070000171661377)
Descriptors: MW 492.94, ALogP 4.06, HBA 6, HBD 3, PSA 114.93, QED 0.32
Compound (SMILES): O=C1/C(=C/c2cccc(F)c2)Cc2cc(O)ccc21
InChIKey: SYWHNFVNAFNPIG-WUXMJOGZSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: Inhibition of recombinant human MAO-A using kynuramine as substrate after 20 mins by fluorescence spectrophotometric analysis
Measurement: IC50 = 458 nM (pChEMBL 6.340000152587891)
Descriptors: MW 254.26, ALogP 3.35, HBA 2, HBD 1, PSA 37.3, QED 0.79
Compound (SMILES): CCN1CCN(C2CCN(C(=O)COc3ccc(-c4ccccc4)cc3)CC2)CC1
InChIKey: BODMOCLWYAYDCG-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Anopheles gambiae
UniProt: Q869C3
Assay: Inhibition of Anopheles gambiae recombinant AChE1 G122S mutant using acetylthiocholine iodide as substrate by Ellman assay
Measurement: IC50 = 1300 nM (pChEMBL 5.889999866485596)
Descriptors: MW 407.56, ALogP 3.36, HBA 4, HBD 0, PSA 36.02, QED 0.74
Compound (SMILES): Cc1cc(-c2ncc(CNC(=O)c3ccc4c(c3)C(=O)c3ccccc3-4)cc2F)ccn1
InChIKey: STHPNWUOZLNVMR-UHFFFAOYSA-N
Target: Protein-serine O-palmitoleoyltransferase porcupine (SINGLE PROTEIN), Mus musculus
UniProt: Q9JJJ7
Assay: Inhibition of porcupine in mouse L Wnt3A cells co-cultured with HEK293 cells after 48 hrs by Super-top flash reporter gene assay
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 423.45, ALogP 4.73, HBA 4, HBD 1, PSA 71.95, QED 0.45
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3c(F)cc(F)cc3F)CC2)C1
InChIKey: WWYPEOYWKHWVHB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Inhibition of human N-terminal His-tagged JAK1 catalytic domain (837 to 1142 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 <= 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 520.52, ALogP 3.47, HBA 7, HBD 1, PSA 106.73, QED 0.43
Compound (SMILES): O=C(Nc1ccc(OC(F)(F)F)cc1)c1cc(-c2cncnc2)ccc1F
InChIKey: ORAYKFFRUFWWLX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Radiometric Filter-Binding Assay: For determination of ABL kinase activity, the radiometric filter-binding assay was used. The assay was performed by mixing 10 μL of the compound pre-diluted with 10 μL of ATP (20 M ATP with 0.1 μCi [γ-33P]-ATP) with the phospho-acceptor peptide poly[Ala6Glu2LysHBr5Tyr1]=polyAEKY) in 20 mM Tris/HCl pH 7.5, 1 mM DTT, 10 mM MgCl2, 0.01 mM Na3VO4, 50 mM NaCl. 10 μL of enzyme (ranging between 5 nM to 20 nM) was added to initiate the reaction. Pre-incubation of enzyme with compounds (when stated) was performed by exposing the enzyme to compounds prior to addition of the substrate mixture (ATP and/or peptide substrate). After 15 min at room temperature, the reaction was stopped by the addition of 50 μL 125 mM EDTA, and the peptide-bound 33P separated on filter-plates (PVDF or MAIP; Millipore, Volketswil, Switzerland) prepared according to the manufacturer's instructions. Filter-plates were washed 3× with 0.5% H3PO4, followed by addition of 30 μL scintillation cocktail (Microscint, Perkin Elmer) per well and then analysed in a TopCount NXT scintillation counter (Perkin Elmer).
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 377.3, ALogP 4.43, HBA 4, HBD 1, PSA 64.11, QED 0.68
Compound (SMILES): CN[C@@H](C)C(=O)NC(Cc1ccc(C#Cc2ccc(C(=O)NC3C[C@@H](C(=O)NC4CCCc5ccccc54)N(C(=O)[C@@H](NC(=O)[C@H](C)NC)C(C)(C)C)C3)cc2)cc1)C(=O)N1C[Si](C)(C)C[C@H]1C(=O)NC1CCCc2ccccc21
InChIKey: KYKMGAUMLNWLEE-NEEMMFLOSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: Homogeneous Time Resolved Fluorescence (HTRF) Assay: Assays were performed in black, flat-bottom, 384-well plates. The final assay volume was 50 μL prepared from additions of His-BIR3 (241-356, XIAP), fluorescein labeled SMAC peptide, and test compounds in assay buffer consisting of 20 mM sodium phosphate, 1 mM EDTA, 50 mM NaCl, 50 μg/ml BSA, and 0.05% PLURONIC® F68. The reaction was incubated at room temperature for 60 minutes, following which 10 μl of mouse anti-6×His-terbium labeled Fab (Medarex, Cisbio) was added to the reaction (40 μl) for an additional 30 minute incubation. The HTRF signal, ratio of fluorescence intensities at emission wavelengths for fluorescein acceptor (520 nm) and terbium donor (615 nm), the 520/615 ratio, generated by the reaction was then measured on the Envision Plate Reader Inhibition data were calculated from the 520/615 ratio generated by the no protein control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay was 1 nM N-His-BIR3(241-356, XIAP), 5 nM fluorescein labeled SMAC peptide, 0.25 nM anti-His-Tb-Fab, and 0.1% DMSO.
Measurement: IC50 = 103 nM (pChEMBL 6.989999771118164)
Descriptors: MW 1104.48
Compound (SMILES): CCn1cc(-c2nc3cnc(N4CCN(C)CC4)cc3n2Cc2ccccc2)cc(C)c1=O
InChIKey: ZNVWETVTURYNSC-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 μM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (LysS, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 μl of the mix is added to the compound dilutions. Subsequently, 6 μl of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 442.57, ALogP 3.39, HBA 7, HBD 0, PSA 59.19, QED 0.47
Compound (SMILES): COc1n[nH]c2cc(NC(=O)NC(C)c3ccc(F)c(F)c3)ncc12
InChIKey: OYVHZYCPPUKGTM-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 77.9 nM (pChEMBL 7.110000133514404)
Descriptors: MW 347.33, ALogP 3.13, HBA 4, HBD 3, PSA 91.93, QED 0.68
Compound (SMILES): O=C(Cc1cccc(C(F)(F)F)c1Cl)NC1COc2nnccc21
InChIKey: WWMREKOKVHDKDR-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: In Vitro Assay: Cell line generation was performed in general according to established molecular cloning protocols. Specifically, RNA was extracted from human whole blood using the Qiagen RNeasy kit (Qiagen, CH) according to the manufacturer's instructions. Subsequently cDNA was made (Superscript II, Invitrogen AG, CH) and the human P2X7 gene (genbank ref. BC011913) was amplified with the following primers: ATCGCGGCCGCTCAGTAAGGACTCTTGAAGCCACT and CGCCGCTAGCACCACCATGCCGGCCTGCTGCAGCTGCA. The amplified sequence was subsequently ligated into a pcDNA3.1 (+) NotI, NheI digested plasmid. Human embryonic kidney (HEK) cells (ATCC CRL-1573, Manassas, Va., USA) were transfected with the pcDNA3.1 (+). hP2X7 plasmid using lipofectamine 2000 (Invitrogen AG, CH) according to the manufacturer's instructions. Following a 24 h exposure to DNA, cells were trypsinized and re-seeded at low density in the presence of 250 ug Geneticin. Geneticin resistant cells were then selected during two consecutive rounds of cloning by serial limiting dilution with visual inspection.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 357.72, ALogP 2.94, HBA 4, HBD 1, PSA 64.11, QED 0.92
Compound (SMILES): COc1cnc2c(Nc3ccc(F)c([C@]4(CF)C[C@@H](C(F)(F)F)OC(N)=N4)c3)nccc2n1
InChIKey: PCNDBEIQPLJGIC-IFXJQAMLSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE FRET Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 468.39, ALogP 3.75, HBA 8, HBD 2, PSA 107.54, QED 0.55
Compound (SMILES): COc1cccc(NC(=O)c2ccc(NC(=O)N3Cc4ccccc4C3)cc2)c1
InChIKey: PCLCEKFPSJNYRY-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 = 99.9 nM (pChEMBL 7)
Descriptors: MW 387.44, ALogP 4.5, HBA 3, HBD 2, PSA 70.67, QED 0.69