lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nccc(-c2ccc(C[C@H](NC(=O)[C@@H]3Cc4cc5c(cc4CN3C(=O)N[C@H](C)c3ccccc3)O[C@@H](c3ccc(OCc4ccc(Cl)cc4Cl)cc3)CO5)C(=O)O)cc2)c1C
InChIKey: LRDYJGIQSFXRJF-HDKDHRJNSA-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 = 133 nM (pChEMBL 6.880000114440918)
Descriptors: MW 885.85, ALogP 9.77, HBA 7, HBD 3, PSA 139.32, QED 0.11
Compound (SMILES): CC(O)[C@H]1O[C@@H](n2cnc3c(OCc4ccccc4)nc(NC(=O)c4ccccc4)nc32)[C@](C)(F)[C@@H]1O
InChIKey: RNRUCLGNYQYKGV-GGOODJTESA-N
Target: RNA-directed RNA polymerase (SINGLE PROTEIN), Hepacivirus hominis
UniProt: O39930
Assay: Replicon Assay: To measure cell-based anti-HCV activity of selected compounds of the present invention, replicon cells were seeded at 5000 cells/well in 96-well collagen I-coated Nunc plates in the presence of the test compound. Various concentrations of test compound, typically in 10 serial 2-fold dilutions, were added to the assay mixture, with the starting concentration ranging from 250 μM to 1 μM. The final concentration of dimethylsulfoxide was 0.5%, fetal bovine serum was 5%, in the assay media. Cells were harvested on day 3 by the addition of 1× cell lysis buffer (Ambion cat #8721). The replicon RNA level was measured using real time PCR (Taqman assay). The amplicon was located in 5B. The PCR primers were: 5B.2F, ATGGACAGGCGCCCTGA (SEQ ID. NO. 1); 5B.2R, TTGATGGGCAGCTTGGTTTC (SEQ ID. NO. 2); the probe sequence was FAM-labeled CACGCCATGCGCTGCGG (SEQ ID. NO. 3). GAPDH RNA was used as endogenous control and was amplified in the same reaction as NS5B (multiplex PCR) using primers and VIC-labeled probe recommended by the manufacturer (PE Applied Biosystem). The real-time RT-PCR reactions were run on ABI PRISM 7900HT Sequence Detection System using the following program: 48° C. for 30 minutes, 95° C. for 10 minutes, 40 cycles of 95° C. for 15 sec, 60° C. for 1 minute.
Measurement: EC50 = 14000 nM (pChEMBL 4.849999904632568)
Descriptors: MW 507.52, ALogP 3.02, HBA 9, HBD 3, PSA 131.62, QED 0.35
Compound (SMILES): O=C1C(N2CC[C@H](c3ccc(O)cc3)[C@@H](F)C2)CCN1Cc1ccc(F)c(F)c1
InChIKey: GEARHVGBHFRSBG-HXOQZHHGSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q00960
Assay: Radioligand Binding Assay: To perform the competition binding assay, thawed membrane homogenate was added to each well of a 96-well plate (20 ug/well). The experimental compounds were serially diluted in 100% DMSO and added to each row of the assay plate to achieve desired compound concentrations, keeping the DMSO concentration in the assay plate at 1.33% of the final reaction volume. Next, 3H Ro 25-6981 (4 nM) was added to the assay plate. After incubation for 1 hr at room temperature, the membrane bound radioligand was harvested on to GF/B filter plates (treated with 0.5% PEI for 1 hr at room temperature). The filter plates were dried at 50° C. for 20 mins, incubated with microscint 20 for 10 minutes and finally, the counts were read on TopCount (Perkin Elmer). Non-specific binding was determined using MK-0657 (the preparation of this compound is described as example 1 in WO 2004 108705 (40 uM).
Measurement: Ki = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 404.43, ALogP 3.6, HBA 3, HBD 1, PSA 43.78, QED 0.85
Compound (SMILES): O=c1[nH]c(N2CCN(c3cccc(C(F)(F)F)c3)CC2)nc(N[C@@H]2CCCNC2)c1-c1nc2ccccc2s1
InChIKey: GXGDJWDOGDMAMZ-GOSISDBHSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IMAP technology (Molecular Devices): A 20 ul reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 uM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 ul of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 555.63, ALogP 4.56, HBA 8, HBD 3, PSA 89.18, QED 0.33
Compound (SMILES): COC(=O)c1cc([C@H](Nc2nc3cc(F)ccc3s2)P(=O)(OC)OC)ccc1O
InChIKey: XLRYJJRTHSPBQO-MRXNPFEDSA-N
Target: 3-hydroxyacyl-CoA dehydrogenase type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99714
Assay: ABAD Enzymatic Activity Assay: The assay for the inhibition of reduction of S-acetoacetyl-CoA (SAAC) by ABAD was carried out with ABAD (418 ng/ml), SAAC (172 μM), NADH (102 μM), and different concentrations of inhibitors (from 0 to 1000 μM) in 93 mM potassium phosphate buffer (pH 7.3). Before the assay, all the assay components except SAAC were pre-incubated for 5 minutes, and the reaction started with the addition of SAAC into the reaction mix. The reaction was carried out for a total 6 minutes at room temperature under steady-state conditions, and the decrease of NADH absorbance at 340 nm was determined every 10 seconds.
Measurement: Ki = 91100 nM (pChEMBL 4.039999961853027)
Descriptors: MW 440.39, ALogP 4.52, HBA 9, HBD 2, PSA 106.98, QED 0.41
Compound (SMILES): CC(C)Nc1cc(-n2ccc3cc(CNC(=O)C4CC4)cnc32)ncc1C(=O)NC[C@@H](F)C(C)(C)O
InChIKey: YRCMXTKNSXAIGF-OAQYLSRUSA-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 = 13.5 nM (pChEMBL 7.869999885559082)
Descriptors: MW 496.59, ALogP 3.11, HBA 7, HBD 4, PSA 121.17, QED 0.34
Compound (SMILES): Cc1cc(S(=O)(=O)Nc2c(C)nn(C)c2C)ccc1Br
InChIKey: LCSQPTBRRXPOOJ-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase (SINGLE PROTEIN), Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
UniProt: Q388H8
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 5400 nM (pChEMBL 5.269999980926514)
Descriptors: MW 358.26, ALogP 2.91, HBA 4, HBD 1, PSA 63.99, QED 0.92
Compound (SMILES): CC(C)(C)/C=C\c1cc(Nc2nccc(C(F)(F)F)n2)cc(-c2cnc([C@@]3(O)CC[C@H](C(=O)O)C(C)(C)C3)s2)c1
InChIKey: PLWTXUHCUFCTBM-TZSNSEKVSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSYK fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-SYK (Carna Biosciences #08-176) (5 pM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 uM final concentration). Final volume of the reaction was 10 uL. Phosphorylation of the peptide was allowed to proceed for 45' at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5, 40 mM EDTA, 0.01% tween 20. Final volume of the quenching solution was 10 uL. The resulting HTRF signal was measured after 30 minutes on an EnVision (Perkin-Elmer) reader using a time-resolved fluorescence protocol.
Measurement: IC50 = 16.84 nM (pChEMBL 7.769999980926514)
Descriptors: MW 574.67, ALogP 7.52, HBA 7, HBD 3, PSA 108.23, QED 0.28
Compound (SMILES): CN1CCCC[C@H](c2ccc(C#Cc3ccccc3)cn2)C1=O
InChIKey: IUDGZOINZXJCGK-GOSISDBHSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Calcium Mobilization Assay: A monoclonal HEK-293 cell line stably transfected with a cDNA encoding for the human mGlu5a receptor was generated; for the work with mGlu5 Positive Allosteric Modulators (PAMs), a cell line with low receptor expression levels and low constitutive receptor activity was selected to allow the differentiation of agonistic versus PAM activity. Cells were cultured according to standard protocols (Freshney, 2000) in Dulbecco's Modified Eagle Medium with high glucose supplemented with 1 mM glutamine, 10% (vol/vol) heat-inactivated bovine calf serum, Penicillin/Streptomycin, 50 mg/ml hygromycin and 15 ug/ml blasticidin (all cell culture reagents and antibiotics from Invitrogen, Basel, Switzerland).About 24 hrs before an experiment, 5x104 cells/well were seeded in poly-D-lysine coated, black/clear-bottomed 96-well plates. The cells were loaded with 2.5 uM Fluo-4AM in loading buffer (1xHBSS, 20 mM HEPES) for 1 hr at 37° C. and washed five times with loading buffer.
Measurement: EC50 = 265 nM (pChEMBL 6.579999923706055)
Descriptors: MW 304.39, ALogP 3.21, HBA 2, HBD 0, PSA 33.2, QED 0.76
Compound (SMILES): CCNc1cc(-n2ccc3cc(Cl)cnc32)ncc1C(=O)NC[C@@H](F)C(C)(C)O
InChIKey: QAZHCVDQTALKJV-MRXNPFEDSA-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 = 7.1 nM (pChEMBL 8.149999618530273)
Descriptors: MW 419.89, ALogP 3.34, HBA 6, HBD 3, PSA 92.07, QED 0.55
Compound (SMILES): CC1(C)CCOc2cc(NC(=O)N3CC=C(c4ncc([C@H](O)CO)cc4F)CC3)ccc21
InChIKey: VMMSBLJVBCJFKR-HXUWFJFHSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Capsaicin-Based Assay: Two days prior to performing this assay, cells are seeded in poly-D-lysine-coated 96-well clear-bottom black plates (50,000 cells/well) in growth media containing 5 μM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4, and cells are loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final). After one hour, the cells are washed twice with 0.2 mL of wash buffer and resuspended in 0.1 mL of wash buffer. The plates are transferred to a FLIPR for assay. 50 μL of test compound diluted with assay buffer (1× Hank's Balanced Salt Solution containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4) are added to the cell plates and incubated for 2 min. The final concentration of the compound is adjusted to range from about 50 picoM to about 3 μM. Human TRPV1 is activated by the addition of 50 μL of capsaicin (400 nM), and the plates are incubated for an additional 3 min.
Measurement: IC50 = 55.1 nM (pChEMBL 7.260000228881836)
Descriptors: MW 441.5, ALogP 3.63, HBA 5, HBD 3, PSA 94.92, QED 0.67
Compound (SMILES): COc1ccc([C@H]2C[C@@H]2COc2cc(NCCc3nccs3)c(=O)n(C)n2)nc1
InChIKey: JHBVDHRPLCUOCD-HIFRSBDPSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product #R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215).
Measurement: Ki = 10.3 nM (pChEMBL 7.989999771118164)
Descriptors: MW 413.5, ALogP 2.48, HBA 9, HBD 1, PSA 91.16, QED 0.58
Compound (SMILES): C[C@]1(CC(=O)O)C[C@H](c2cccc(Cl)c2)[C@@H](c2ccc(Cl)cc2)N(c2ccncn2)C1=O
InChIKey: DMCGIUZKUVSMAM-ZFJSRUIDSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Homogenous Time-Resolved Fluorescence Assay (HTRF1 Assay): The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1xPBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-S-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag-TrxA-6-His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography.Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3.
Measurement: IC50 = 340 nM (pChEMBL 6.46999979019165)
Descriptors: MW 470.36, ALogP 5.53, HBA 4, HBD 1, PSA 83.39, QED 0.53
Compound (SMILES): COC(=O)N1CCC(c2cnc(N)c(-c3ccc(C(=O)N[C@H](CO)c4ccccc4)cc3)c2)CC1
InChIKey: WRZSCHMPRIUUGL-XMMPIXPASA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 (20 pM) Kinase Assay: Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 20 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.02 nM ERK2, 400 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 4 hours at ambient temperature. Reaction was terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 20 ug/mL of AlphaScreen Protein A Acceptor Beads, 20 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1:1000 dilution phospho-EGF Receptor (Thr669) antibody (Cat #8808, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values were determined using a four-parameter fit.
Measurement: IC50 = 15.8 nM (pChEMBL 7.800000190734863)
Descriptors: MW 474.56, ALogP 3.74, HBA 6, HBD 3, PSA 117.78, QED 0.5
Compound (SMILES): CC(C)CN(Cc1ccc(N2CC[C@@H](O)C2)cc1)S(=O)(=O)Cc1ccccc1
InChIKey: CONJPXFUCYVLQH-JOCHJYFZSA-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 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 402.56, ALogP 3.25, HBA 4, HBD 1, PSA 60.85, QED 0.74
Compound (SMILES): COCCCn1nc(C)c2c(Cl)cc([C@@H](C)N(C(=O)[C@H]3CNCCN3)C3CC3)cc21
InChIKey: LGEJHNUSHWOFFH-DNVCBOLYSA-N
Target: Renin (SINGLE PROTEIN), Homo sapiens
UniProt: P00797
Assay: Inhibitory Assay: A substrate of synthetic peptide (Nma-KHPFHLVIHK(Dnp)-NH2) and test compound were mixed, and fluorescence intensity was assayed using a fluorophotometer before staring an enzymatic reaction (exciting wavelength: 340 nm, measuring wavelength: 460 nm). Recombinant human renin was added and the mixture was incubated at 37° C. for 1 hour, and the fluorescence intensity was measured after the reaction using a fluorophotometer (exciting wavelength: 340 nm, measuring wavelength: 460 nm). Renin activity was evaluated on the ground of fluorescence intensity which was obtained by deduction of the intensity before the reaction from the intensity after the reaction, and 50% inhibitory concentration (IC50) was calculated from renin activities under the existence of various concentration of the test compound.
Measurement: IC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 433.98, ALogP 2.65, HBA 6, HBD 2, PSA 71.42, QED 0.63
Compound (SMILES): COCCCOc1cc([C@@H](C)N(C(=O)[C@H]2CNCCO2)C2CC2)ccc1OC(F)F
InChIKey: ZUFNFHGTAJJORS-AUUYWEPGSA-N
Target: Renin (SINGLE PROTEIN), Homo sapiens
UniProt: P00797
Assay: Inhibitory Assay: A substrate of synthetic peptide (Nma-KHPFHLVIHK(Dnp)-NH2) and test compound were mixed, and fluorescence intensity was assayed using a fluorophotometer before staring an enzymatic reaction (exciting wavelength: 340 nm, measuring wavelength: 460 nm). Recombinant human renin was added and the mixture was incubated at 37° C. for 1 hour, and the fluorescence intensity was measured after the reaction using a fluorophotometer (exciting wavelength: 340 nm, measuring wavelength: 460 nm). Renin activity was evaluated on the ground of fluorescence intensity which was obtained by deduction of the intensity before the reaction from the intensity after the reaction, and 50% inhibitory concentration (IC50) was calculated from renin activities under the existence of various concentration of the test compound.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 428.48, ALogP 2.74, HBA 6, HBD 1, PSA 69.26, QED 0.55
Compound (SMILES): C[C@@H](NC(=O)c1cc(C(=O)N2CCC[C@@H]2C(F)(F)F)c2n1CCOC2)c1ccc(F)cc1
InChIKey: DBYYWKZVBIEKFG-BFUOFWGJSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: TASK-1 Inhibiition Assay: Human TASK-1 channels were expressed in Xenopus oocytes. For this purpose, oocytes were isolated from Xenopus laevis and defoliculated. Subsequently, TASK-1-encoding RNA synthesized in vitro was injected into the oocytes. After two days of TASK-1 protein expression, TASK-1 currents were measured by two-microelectrode voltage clamp. Data were acquired and analyzed using a TEC-10cx amplifier (NPI Electronic, Tamm, Germany) connected to an ITC-16 interface (Instrutech Corp., Long Island, USA) and Pulse software (HEKA Elektronik, Lambrecht, Germany). Oocytes were clamped to -90 mV and TASK-1 mediated currents were measured during 500 ms voltage pulses to 40 mV. Oocytes were continuously superfused with ND96 buffer containing 96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 5 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES; pH adjusted to 7.4 with sodium hydroxide). All experiments were performed at room temperature.
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 453.44, ALogP 3.82, HBA 4, HBD 1, PSA 63.57, QED 0.72
Compound (SMILES): Cc1nn(C)c(C)c1NS(=O)(=O)c1ccc(CNc2ccc(C(C)C)cc2)cc1
InChIKey: VRXYUSDGVXZTAN-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase (SINGLE PROTEIN), Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
UniProt: Q388H8
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 9700 nM (pChEMBL 5.010000228881836)
Descriptors: MW 412.56, ALogP 4.57, HBA 5, HBD 2, PSA 76.02, QED 0.6
Compound (SMILES): Cc1cc(-c2nc(-c3ccc(OC(F)(F)F)c(C#N)c3)n3c2CCO[C@H](CF)C3)ccn1
InChIKey: BMFSKNNNHYWJQB-QGZVFWFLSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: [35S]GTPgammaS Binding Assay: The frozen cell membrane fraction prepared as described above was thawed before use, and the resultant was diluted with a buffer for a binding assay (final concentrations: 20 mM HEPES, 100 mM NaCl, 1 mM MgCl2, 3 uM GDP, 300 ug/mL saponin, 0.1% BSA). The compound of each example was added to a cell membrane fraction containing 1.8 to 3 ug/assay of membrane protein on a plate, followed by incubation at room temperature for 30 minutes. Thereafter, with glutamic acid (in a final concentration of 10 uM) added thereto, incubation was performed at room temperature for 15 minutes, and thereafter, [35S]GTPγS (in a final concentration of 0.8 kBq) and 588 ug WGA-SPA beads were added thereto, followed by incubation at room temperature for 1 hour. After the incubation, the plate was subjected to the centrifugal separation at 2,500 rpm and room temperature, and then, membrane cell binding radioactivity was measured with a top count. A [35S]GTPγS binding amount obtained in the presence of glutamic acid was defined as specific binding. On the basis of ratios of inhibiting the specific binding at various concentrations of the compounds of the respective examples, inhibition curves were obtained. Concentrations of the compounds of the respective examples at which the specific [35S]GTPγS binding amount was suppressed by 50% (IC50 values) were calculated on the basis of the inhibition curves.
Measurement: IC50 = 3.8 nM (pChEMBL 8.420000076293945)
Descriptors: MW 446.4, ALogP 4.6, HBA 6, HBD 0, PSA 72.96, QED 0.55
Compound (SMILES): O=C(Nc1ccc(C(F)(F)F)c(Cl)c1)N1CCC(F)(c2ncc([C@H](O)CO)cc2F)CC1
InChIKey: QHDMAJLOOMYSQM-MRXNPFEDSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Capsaicin-Based Assay: Two days prior to performing this assay, cells are seeded in poly-D-lysine-coated 96-well clear-bottom black plates (50,000 cells/well) in growth media containing 5 μM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4, and cells are loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final). After one hour, the cells are washed twice with 0.2 mL of wash buffer and resuspended in 0.1 mL of wash buffer. The plates are transferred to a FLIPR for assay. 50 μL of test compound diluted with assay buffer (1× Hank's Balanced Salt Solution containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4) are added to the cell plates and incubated for 2 min. The final concentration of the compound is adjusted to range from about 50 picoM to about 3 μM. Human TRPV1 is activated by the addition of 50 μL of capsaicin (400 nM), and the plates are incubated for an additional 3 min.
Measurement: IC50 = 14.3 nM (pChEMBL 7.840000152587891)
Descriptors: MW 479.83, ALogP 4.41, HBA 4, HBD 3, PSA 85.69, QED 0.57
Compound (SMILES): CC(C)(C)OC(=O)N1CCCC1C(=O)N1CC[C@@]2(C)c3cccc(O)c3C[C@@H]1C2(C)C
InChIKey: UNYXRNUZCSNQSF-ZESTXYGFSA-N
Target: 11-beta-hydroxysteroid dehydrogenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28845
Assay: Homogeneous Time-Resolved Fluorescence (HTRF): In vitro inhibition of 11beta -HSD1 by test compounds was determined with HTRF (Homogeneous Time-Resolved Fluorescence) technology (cisbio international, France) detecting cortisol generated from cortisterone by human liver microsomes. Briefly, compounds were incubated for 1 hour at 37 C. in Tris buffer (20 mM tris, 5 mM EDTA, pH 6.0) containing NADPH (200 uM) and cortisone (80 nM). Cortisol generated in the reaction is then detected with a competitive immunoassay, involving two HTRF conjugates: cortisol linked to XL665 and anti-cortisol antibody labeled with Europium cryptate. The incubation period for detection reaction was typically 2 hours. The amount of cortisol is determined by reading the time-resolved fluorescence of the wells (Ex 320/75 nm; Em 615/8.5 nm and 665/7.5 nm). The ratio of the two emission signals is then calculated (Em665*10000/Em615).
Measurement: IC50 = 2656 nM (pChEMBL 5.579999923706055)
Descriptors: MW 428.57, ALogP 4.23, HBA 4, HBD 1, PSA 70.08, QED 0.73
Compound (SMILES): CC1(C)c2cc(OC[C@@H](O)[C@H](O)CO)ccc2C(=O)c2c1oc1cc(OC(=O)NCc3ccccc3)ccc21
InChIKey: KQOHVFQJKPFIER-DNQXCXABSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: ALK-inhibiting activity was measured by following an activity of inhibiting phosphorylation by biotinylated peptide (EGPWLEEEEEAYGWMDF). For the detection of phosphorylation of the biotinylated peptide, time-resolved fluorescence measurement was performed using an anti-phosphorylated tyrosine antibody labeled with europium cryptate and streptavidin conjugated to XL665, i.e., an allophycocyanin derivative.
Measurement: IC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 531.56, ALogP 3.68, HBA 8, HBD 4, PSA 138.46, QED 0.27
Compound (SMILES): COC(=O)N[C@H](C(=O)N[C@H](c1ccc([C@@H](CO)N(CC(C)C)S(=O)(=O)c2ccc(N)cc2)s1)C1CC1)C(c1ccccc1)c1ccccc1
InChIKey: SDGWVQCQKMVUIE-DCCHFUKUSA-N
Target: Gag-Pol polyprotein (SINGLE PROTEIN), Human immunodeficiency virus type 1 group M subtype B (isolate HXB2)(HIV-1)
UniProt: P04585
Assay: Inhibition Assay: The inhibition of WT HIV-1 protease was studied using the reaction of the protease (expressed in Eschericia coli) with a peptide substrate [Val-Ser-Gln-Asn-(betanaphthyl)Ala-Pro-Ile-Val (SEQ ID NO:1)]. Compounds were tested using Procedure A. In Procedure A, the test compound was first preincubated with the enzyme in assay buffer (50 mM sodium acetate, pH 5.5, 100 mM NaCl, and 0.1% BSA) for 30 minutes at room temperature. Substrate was then added to 400 micromolar in a total volume of 80 microliters containing 10 picomolar HIV-1 protease and the reaction was incubated for 1 hour at 30° C. The reaction was quenched with the addition of 120 microliters of 10% phosphoric acid. The product formation was determined after separation of product and substrate on a Zorbax Eclipse XDB-C18 column (Agilent Technologies, Santa Clara, Calif.) connected to an Agilent 1100 high performance liquid chromatography system with fluorescence detection (excitation 270 nanometer and emission 330 nanometer).
Measurement: IC50 = 3.98 nM (pChEMBL 8.399999618530273)
Descriptors: MW 704.92, ALogP 5.83, HBA 8, HBD 4, PSA 151.06, QED 0.11
Compound (SMILES): NC[C@@H](NC(=O)c1ccc(-c2nc([C@@H]3CC[C@@H](O)[C@H](F)C3)cnc2N)cc1F)c1cc(F)cc(Br)c1
InChIKey: NTYBOGUXDZKPQH-UZWLDWCWSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 (20 pM) Kinase Assay: Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 20 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.02 nM ERK2, 400 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 4 hours at ambient temperature. Reaction was terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 20 ug/mL of AlphaScreen Protein A Acceptor Beads, 20 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1:1000 dilution phospho-EGF Receptor (Thr669) antibody (Cat #8808, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values were determined using a four-parameter fit.
Measurement: IC50 = 0.0139 nM (pChEMBL 10.859999656677246)
Descriptors: MW 564.41, ALogP 4.16, HBA 6, HBD 4, PSA 127.15, QED 0.36
Compound (SMILES): CN1CCN(C(=O)Nc2cnc3ccc(N4CCC[C@@H]4c4cc(F)ccc4F)nn23)CC1
InChIKey: RWIABVRWALKQEZ-GOSISDBHSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: ELISA Assay: An enzyme-linked immunosorbant assay (ELISA) was used to assess TrkA kinase activity in the presence of inhibitors. Immulon 4HBX 384-well microtiter plates (Thermo part #8755) were coated with a 0.025 mg/mL solution of poly (Glu, Ala, Tyr; 6:3:1; Sigma P3899). Various concentrations of test compound, 2.5 nM TrkA (Invitrogen Corp., histidine-tagged recombinant human TrkA, cytoplasmic domain), and 500 uM ATP were incubated for 25 minutes at ambient temperature in the coated plates while shaking. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The reaction mixture was removed from the plate by washing with PBS containing 0.1% (v/v) Tween 20. The phosphorylated reaction product was detected using 0.2 ug/mL of a phosphotyrosine specific monoclonal antibody (clone PY20) conjugated to horseradish peroxidase in conjunction with the TMB Peroxidase Substrate System (KPL).
Measurement: IC50 = 53.2 nM (pChEMBL 7.269999980926514)
Descriptors: MW 441.49, ALogP 3.13, HBA 6, HBD 1, PSA 69.01, QED 0.68
Compound (SMILES): C[C@]12CCCN1CC1=NNC(=O)c3cccc4[nH]c2c1c34
InChIKey: YDYWCKXGGCVENO-MRXNPFEDSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP-1 Enzymatic Assay: PARP-1 enzymatic assay was conducted using a method modified from HT F Homogeneous PARP Inhibition Assay Kit (Trevigen). 8.8 nM PARP-1 was pre-incubated with different concentrations of compounds in a buffer containing 100 mM Tris-HCl pH 8.0, 100 mM NaCl, 20 mM MgCl2, and 1% DMSO for 30 min at RT. The auto-PARylation reaction was initiated by addition of 500 nM NAD and 20 ng/ul activated DNA (Sigma) and incubated at RT for 40 min. The remaining NAD was detected by incubation with cycling assay solution containing 1% ethanol, 0.30 U/ml alcohol dehydrogenase, 25 uM resazurin, and 0.25 U/ml diaphorase for 50 min at RT. The concentration of NAD is proportional to the fluorescence signal at Ex 540 nm/Em 590 nm.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 280.33, ALogP 1.94, HBA 3, HBD 2, PSA 60.49, QED 0.77
Compound (SMILES): CC(C)(C)C[C@H]1N[C@@H](C(=O)NC2CCC(OC(=O)C(C)(C)N)CC2)[C@H](c2cccc(Cl)c2F)[C@@]12C(=O)Nc1cc(Cl)ccc12
InChIKey: CCBWZZYXLQJFDQ-HQDKQNMLSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Fluorescence-Polarization Binding Assay: The binding affinity of the MDM2 inhibitors was determined using an optimized, sensitive and quantitative fluorescence polarization-based (FP-based) binding assay using a recombinant human His-tagged MDM2 protein (residues 1-118) and a fluorescently tagged p53-based peptide.The design of the fluorescence probe was based upon a previously reported high-affinity p53-based peptidomimetic compound (5-FAM-beta Ala-beta Ala-Phe-Met-Aib-pTyr-(6-Cl-LTrp)-Glu-Ac3c-Leu-Asn-NH2 (SEQ ID NO: 1)) Garcia-Echeverria et al., J. Med. Chem. 43: 3205-3208 (2000)). This tagged peptide is called PMDM6-F. The Kd value of PMDM6-F with the recombinant MDM2 protein was determined from the saturation curve. MDM2 protein was serially double diluted in a Dynex 96-well, black, round-bottom plate, and the PMDM6-F peptide was added at 1 nM concentration. The assay was performed in the buffer: 100 mM potassium phosphate, pH 7.5; 100 ug/mL bovine gamma globulin; 0.02% sodium azide, 0.01% Triton X-100).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 647.62, ALogP 5.59, HBA 6, HBD 4, PSA 122.55, QED 0.3
Compound (SMILES): O=C(Nc1ccc(Cl)c(C(F)(F)F)c1)N1CC=C(c2ncc([C@H](O)CO)cc2Cl)CC1
InChIKey: FMTUNEXVOUHOHA-QGZVFWFLSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Capsaicin-Based Assay: Two days prior to performing this assay, cells are seeded in poly-D-lysine-coated 96-well clear-bottom black plates (50,000 cells/well) in growth media containing 5 μM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4, and cells are loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final). After one hour, the cells are washed twice with 0.2 mL of wash buffer and resuspended in 0.1 mL of wash buffer. The plates are transferred to a FLIPR for assay. 50 μL of test compound diluted with assay buffer (1× Hank's Balanced Salt Solution containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4) are added to the cell plates and incubated for 2 min. The final concentration of the compound is adjusted to range from about 50 picoM to about 3 μM. Human TRPV1 is activated by the addition of 50 μL of capsaicin (400 nM), and the plates are incubated for an additional 3 min.
Measurement: IC50 = 225.8 nM (pChEMBL 6.650000095367432)
Descriptors: MW 476.28, ALogP 4.75, HBA 4, HBD 3, PSA 85.69, QED 0.59
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(CCC3CC3)cc1C21NC(N)N([C@H](C)c2ccccc2)C1=O
InChIKey: QQSLGIHTNHTVGI-ZCNXRHOSSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.435): 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). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 ul OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 ul water containing the desired concentration of test compound and 1% DMSO, substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were given to the plate followed by the addition of 10 ul of BACE.
Measurement: IC50 = 81.1 nM (pChEMBL 7.090000152587891)
Descriptors: MW 473.66, ALogP 4.79, HBA 4, HBD 2, PSA 67.59, QED 0.63
Compound (SMILES): O=C(Nc1ccc([C@H]2CNCCO2)cc1)c1cnn(-c2cccc(Cl)c2)n1
InChIKey: DTOFQGJNVRBQCD-GOSISDBHSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding (mouse TAAR1): HEK-293 cells stably expressing mouse TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 μg/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1'000 rpm for 5 min at 4μ C., frozen and stored at −80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14'000 rpm for 20 s. The homogenate was centrifuged at 48'000×g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14'000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at −80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]−(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 0.7 nM, resulting in the binding of approximately 0.5% of the radioligand and a specific binding representing approximately 70% of the total binding. Nonspecific binding was defined as the amount of 3[H]−(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 μM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 μM) in duplicates. The test compounds (20 μl/well) were transferred into a 96 deep well plate (TreffLab), and 180 μl of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 μl of the radioligand 3[H]−(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3×Kd in nM and 500 μl of the membranes (resuspended at 60 μg protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 μl of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 383.84, ALogP 2.83, HBA 6, HBD 2, PSA 81.07, QED 0.72
Compound (SMILES): OC[C@H]1O[C@@H](c2cc(Cc3ccc(C4CCC4)cc3)c(Cl)c3c2OCCC3)[C@H](O)[C@@H](O)[C@@H]1O
InChIKey: AZNJZHUZHCLPNI-TXKDOCKMSA-N
Target: Sodium/glucose cotransporter 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P31639
Assay: SGLT2 Inhibition Assay: For sodium-dependent glucose transport assay, cells expressing hSGLT2 were seeded into a 96-well culture plate at a density of 5×104 cells/well in RPMI medium 1640 containing 10% fetal bovine serum. The cells were used 1 day after plating. They were incubated in pretreatment buffer (10 mM HEPES, 5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl2, and 1 mM MgCl2, pH 7.4) at 37 °C. for 10 min. They were then incubated in uptake buffer (10 mM HEPES, 5 mM Tris, 140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM 14C-nonlabeled AMG pH 7.4) containing 14C-labeled AMG (8 μM) and the inventive compound or dimethyl sulfoxide (DMSO) vehicle at 37 °C. for 2 h. Cells were washed twice with washing buffer (pretreatment buffer containing 10 mM AMG at room temperature) and then the radioactivity was measured using a liquid scintillation counter.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 474.98, ALogP 3.04, HBA 6, HBD 4, PSA 99.38, QED 0.53
Compound (SMILES): CC(=O)C1=C(C)N(c2cccc(C(F)(F)F)c2)C(=O)N(C)[C@@H]1c1ccc(C#N)cc1C(=O)NCC#N
InChIKey: FIMMGUUXADWRJM-JOCHJYFZSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: Materials: Human neutrophil elastase was purchased from Calbiochem (Cat. No.: 324681) and the elastase substrate MeOSuc-Ala-Ala-Pro-Val-AMC from Bachem (Cat. No.: I-1270). All other materials were of the highest grade commercially available.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 uL, of these compound dilutions were mixed with 10 uL 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 uL, substrate solution in assay buffer were added (250 uM 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. Each plate contains wells with a high value control (DMSO+enzyme+substrate) and wells with a low value control (DMSO+inactivated enzyme+substrate). IC50 values were estimated using a sigmoidal concentration response curve with variable slope.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 495.46, ALogP 4.31, HBA 5, HBD 1, PSA 117.3, QED 0.62
Compound (SMILES): N#CC1CCC(Oc2n[nH]c3cc(NC(=O)N[C@H](COC(F)F)c4ccccc4)ncc23)CC1
InChIKey: GLZCNWRHCOIQGN-GSQSYGFSSA-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 = 6.2 nM (pChEMBL 8.210000038146973)
Descriptors: MW 470.48, ALogP 4.52, HBA 6, HBD 3, PSA 124.95, QED 0.44