lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): c1csc(-c2nc(NC3Cc4ccccc4C3)c3ccccc3n2)c1
InChIKey: YIFJXSPQCBCHCI-UHFFFAOYSA-N
Target: Lysosomal acid glucosylceramidase (SINGLE PROTEIN), Homo sapiens
UniProt: P04062
Assay: Inhibition of synthetic recombinant wild type GCase enzyme velaglucerase alfa (unknown origin) at pH 5.9 preincubated for 5 mins followed by addition of 4-Methylumbelliferyl beta-D-glucopyranoside as substrate measured after 30 mins by fluorescence analysis
Measurement: IC50 = 8.2 nM (pChEMBL 8.09000015258789)
Descriptors: MW 343.46, ALogP 4.94, HBA 4, HBD 1, PSA 37.81, QED 0.57
Compound (SMILES): CCOc1cc(N2CCC(CCS(C)(=O)=O)CC2)c(CC)cc1Nc1nccc(-c2c(-c3ccc(OCC)c(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: IWQCCUGBEIGWSH-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human GST-rTEV-tagged IGF1R (957 to 1367 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 823.97, ALogP 8.74, HBA 11, HBD 2, PSA 140.05, QED 0.1
Compound (SMILES): O=C(CO)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(O)c1)C2)Cc1ccccc1
InChIKey: FFBZQZXSTZLCDJ-KOUNCHBCSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Displacement of [3H]-diprenorphine from human mu opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 0.07943 nM (pChEMBL 10.100000381469727)
Descriptors: MW 394.52, ALogP 3.12, HBA 4, HBD 2, PSA 64.01, QED 0.76
Compound (SMILES): Cc1c(-c2ncnc3[nH]c(-c4ccc(C(=O)NCC(F)(F)F)cc4)cc23)cccc1N1CCOc2cc(C3CC3)ccc2C1=O
InChIKey: INQHGIOCJYRMJF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Recombinant Human BTK Inhibition Assay: The inhibition assays were performed using the generic tyrosine kinase assay kit from C is Bio (HTRF KinEASE-TK #62TK0PEB). The assay is based on the principle of fluorescence resonance energy transfer (FRET) between the Europium Cryptate labeled anti-phosphotyrosine antibody and streptavidin labeled fluorophore, XL-665. In an uninhibited kinase reaction the biotinylated peptide substrate (Tk-Biotin) gets phosphorylated at an intermediate tyrosine site which is captured by anti-phosphotyrosine antibody which eventually acts as a FRET donor. The streptavidin labeled XL-665 binds strongly to the Tk-Biotin substrate and act as a FRET acceptor. The resultant FRET signal is measured at 665 nm. A ratio of this specific fluorescence (665 nm) to the background fluorescence (620 nm), conferred by the free antibody, is directly proportional to the kinase activity. Inhibition of the kinase activity by a pharmacological intervention such as a Btk inhibitor of the present disclosure will result into decreased FRET signal.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 611.62, ALogP 6.81, HBA 5, HBD 2, PSA 100.21, QED 0.22
Compound (SMILES): C[C@@H]1CN(c2n[nH]c3cc(NC(=O)N[C@@H](c4ccccc4)C(C)(C)O)ncc23)C[C@H](C)O1
InChIKey: DPRNZWSVFLTVAV-MDOVXXIYSA-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. The final reaction in each well of 25 uL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 uM ATP.
Measurement: IC50 = 0.8 nM (pChEMBL 9.100000381469727)
Descriptors: MW 438.53, ALogP 3.21, HBA 6, HBD 4, PSA 115.4, QED 0.49
Compound (SMILES): Cc1c(N2CCN(c3cnc(C(C)(C)O)cn3)[C@H](C)C2)nnc(Cc2ccccc2)c1CO
InChIKey: MRGOROJMQXXDLJ-QGZVFWFLSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 448.57, ALogP 2.6, HBA 8, HBD 2, PSA 98.5, QED 0.59
Compound (SMILES): CCN(CC(=O)NCC1CN(c2ccc(C(F)(F)F)cc2)CCO1)S(=O)(=O)c1ccc(F)cc1
InChIKey: QQQSONYYTGJIQO-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: Dose Response Assay: Compound plates were resuspended with 100 ul of HBSS/20 mM HEPES/0.005% BSA buffer (Compound Buffer): column 1A-H: buffer/DMSO (bk); column 2A-H: AP-18 (control antagonist for CHOK1 TRPA1 cells); column 1I-P: ATP (control for CHOK1 teton cells); column 2 I-P: 2APB (control antagonist for CHOK1/TRPM8 cells). Growth media was removed from the cell plates (20 ul) and 20 ul of the Replacement Buffer was added followed by addition of 25 ul of diluted dye. All three steps were performed using a Plate Washer BioTek 407. The plates were then incubated for 30' at RT. After incubation, both the cell and compound plates were brought to the FLIPR and 20 ul of the diluted compounds/antagonist/bk were transferred to the cell plates by the FLIPR. Plates were then incubated for 30' at room temperature. After 30' incubation, plates were returned to the FLIPR and 20 ul of 4.5× Cinnamaldehyde was added to the cell plates. During the compound addition as well as agonist addition, fluorescence readings were taken simultaneously from all 384 wells of the cell plate every 1.5 seconds. Five readings were taken to establish a stable baseline, then 20 ul of sample was rapidly (30 ul/sec) and simultaneously added to each well of the cell plate. The fluorescence was continuously monitored before, during and after sample/agonist addition for a total elapsed time of 100 seconds (compound addition) and 120 seconds (agonist addition).
Measurement: IC50 = 226 nM (pChEMBL 6.650000095367432)
Descriptors: MW 503.52, ALogP 2.88, HBA 5, HBD 1, PSA 78.95, QED 0.56
Compound (SMILES): CC[C@@H](C(N)=O)N1CC(CCC(F)(F)F)CC1=O
InChIKey: LJBKVYFUXPAUJV-MQWKRIRWSA-N
Target: Synaptic vesicle glycoprotein 2A (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q02563
Assay: Displacement of [3H]-2-[4-(3-azidophenyl)-2-oxo-1-pyrrolidinyl]butanamide from SV2A levetiracetam binding site in Sprague-Dawley rat cerebral cortex membranes after 120 mins by liquid scintillation counting method
Measurement: Ki <= 1000 nM (pChEMBL 6)
Descriptors: MW 266.26, ALogP 1.44, HBA 2, HBD 1, PSA 63.4, QED 0.82
Compound (SMILES): NC(=O)c1c(-c2ccc(Oc3ccccc3)cc2)nn2c1NCCC2c1cccc(N)c1
InChIKey: LDGHRNHLQSSGCZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Time-Resolved Fluorescence Resonance Energy Transfer Assay: Compounds disclosed herein were tested for inhibition of Btk kinase activity in an assay based on time-resolved fluorescence resonance energy transfer methodology. Recombinant Btk was pre-incubated with the compounds disclosed herein at room temperature for 1 hour in an assay buffer containing 50 mM Tris pH7.4, 10 mM MgCl2, 2 mM MnCl2, 0.1 mM EDTA, 1 mM DT, 20 nM SEB, 0.1% BSA, 0.005% tween-20. The reactions were initiated by the addition of ATP (at the concentration of ATP Km) and peptide substrate (Biotin-AVLESEEELYSSARQ-NH2). After incubating at room temperature for 1 h, an equal volume of stop solution containing 50 mM HEPES pH7.0, 800 mM KF, 20 mM EDTA, 0.1% BSA, Eu cryptate-conjugated p-Tyr66 antibody and streptavidin-labeled XL665 was added to stop the reaction. Plates were further incubated at room temperature for 1 hour, and then the TR-FRET signals (ex337 nm, em 620 nm/665 nm) were read on BMG PHERAstar FS instrument. The residual enzyme activity in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 615 nm to that at 665 nm. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Measurement: IC50 = 360 nM (pChEMBL 6.440000057220459)
Descriptors: MW 425.49, ALogP 4.43, HBA 6, HBD 3, PSA 108.19, QED 0.41
Compound (SMILES): COc1ccc(C(=O)N2CCOC[C@H]2Cc2cccc(-n3nccn3)c2)c(-n2nccn2)c1
InChIKey: MRZOKJFVVJMJLC-LJQANCHMSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Antagonist activity at human OX1R expressed in CHO cells preincubated for 120 mins followed by orexin-A addition by FLIPR assay
Measurement: IC50 = 6.2 nM (pChEMBL 8.210000038146973)
Descriptors: MW 445.48, ALogP 1.94, HBA 9, HBD 0, PSA 100.19, QED 0.45
Compound (SMILES): CCCN(CCC)[C@@H](C)C(=O)Nc1cccc(Cc2n[nH]c(=O)c3c2CCCC3)c1
InChIKey: KSZYOAGICNCFCW-KRWDZBQOSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 1590 nM (pChEMBL 5.800000190734863)
Descriptors: MW 410.56, ALogP 3.69, HBA 4, HBD 2, PSA 78.09, QED 0.66
Compound (SMILES): CCCN(CCC)C(=O)C1=Cc2ccc(C(=O)Nc3cncc(CN(C)C)c3)cc2N=C(N)C1
InChIKey: BDADHWAXNCEEGE-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Pharmacological Assay: For TLR8 and TLR7 activity testing, HEK-Blue human TLR8 or TLR7 cells (Invivogen, San Diego, Calif., USA) are used, respectively. These cells are designed for studying the stimulation of human TLR8 or TLR7 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene is placed under the control of the IFN-b minimal promoter fused to five NF-κB and AP-1-binding sites. Therefore the reporter expression is regulated by the NF-κB promoter upon stimulation of human TLR8 or TLR7 for 20 hours. The cell culture supernatant SEAP reporter activity was determined using Quanti Blue kit (Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple/blue in the presence of alkaline phosphatase.
Measurement: EC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 462.6, ALogP 3.82, HBA 6, HBD 2, PSA 103.92, QED 0.59
Compound (SMILES): Nc1nccc2cc(C[C@H](C(=O)O)N3CC[C@H](NS(=O)(=O)c4cnc(N5CCCC5)s4)C3=O)c(Cl)cc12
InChIKey: SYIZRHNKMPFMEA-ZWKOTPCHSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: Inhibition Assay: The compounds according to the invention are tested in a range of concentrations (4.9 nM to 5 µM final) and deposited in a proportion of 25 µl per well. After the deposition of 50 µl of Spectrozyme 229 substrate (American Diagnostica), at the final concentration of 625 µM, the reaction is triggered by the addition of 25 µl of FIXa (human factor IXa supplied by Enzyme Research Laboratories (ERL)), at the final concentration of 2.5 U/ml. Reading is carried out at 405 nm for 15 min at 37°C. The percentage of inhibition of enzymatic activity (expressed as maximum rate of cleavage of the substrate) is calculated with respect to the enzymatic activity in the absence of inhibitor.
Measurement: IC50 = 84 nM (pChEMBL 7.079999923706055)
Descriptors: MW 565.08, ALogP 2.1, HBA 9, HBD 3, PSA 158.82, QED 0.37
Compound (SMILES): CCCCOC(=O)[C@H](C)NP(=O)(Oc1cccc2ccccc12)O[C@H](CC)[C@H]1O[C@@H](n2cc(F)c3c(N)ncnc32)[C@](C)(O)[C@@H]1O
InChIKey: PQRFBPPNWVABOP-NKZHOLKVSA-N
Target: RNA-directed RNA polymerase (SINGLE PROTEIN), Hepacivirus hominis
UniProt: O39930
Assay: Polymerase Inhibition Assay: The ability of the nucleoside triphosphate derivatives to inhibit the enzymatic activity of the HCV NS5B RNA-dependent RNA polymerase was measured in a radiolabeled nucleotide incorporation assay modified from the method described in International Publication No. WO2002/057287. Briefly, 50 μL reactions containing 20 mM HEPES (pH 7.3), 7.5 mM DTT, 20 units/mL RNasIN, 1 μM each of ATP, GTP, UTP and CTP, 20 μCi/mL [33P]-CTP, 10 mM MgCl2, 60 mM NaCl, 100 μg/ml BSA, 0.021 μM DCoH heteropolymer RNA template and 5 nM NS5B (1b-BKΔ55) enzyme were incubated at room temperature for 1 hour. Assay was terminated by the addition of 500 mM EDTA (50 μL). The reaction mixture was transferred to a Millipore DE81 filter plate and the incorporation of labeled CTP was determined using Packard TopCount.
Measurement: EC50 = 9300 nM (pChEMBL 5.03000020980835)
Descriptors: MW 659.65, ALogP 4.62, HBA 12, HBD 4, PSA 180.28, QED 0.09
Compound (SMILES): COCCCNC(=O)c1ccc(Nc2ncc3c(n2)-c2c(nn(C)c2-c2ccc(-c4cnn(C)c4)cc2)CC3)c(OC)c1
InChIKey: OYQMWWVIYBSJLB-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: DELFIA Assay: The kinase activity is measured by DELFIA assay (dissociation-enhanced lanthanide fluorescence immunoassay, Perkin Elmer). The cytoplasmic kinase domain of human IGF-1R (amino acids 964-1370) is expressed as a fusion protein with a glutathione-S-transferase tag (IGF-1R-GST) in High Five.TM. Cells (Invitrogen). Enzyme activity is measured in the presence of substances and a control substance. Poly-glutamate-tyrosine peptide (pEY, Sigma Aldrich) and biotinylated pEY (bio-pEY) are used as reaction substrates.10 uL of substance in 25% DMSO are mixed with 30 uL of IGF-1R-GST solution (67 mM HEPES pH 7.4, 15 ug/mL pEY, 1.7 ug/mL bio-pEY, 13.3 mM MgCl2, 3.3 mM dithiothreitol, 0.0033% Brij 35, 2 ng IGF-1R-GST) in 96-well plates. The reactions are started with 10 uL of a 750 uM ATP solution. After 40 min at RT the reactions are stopped with 50 .mu.L of stop solution (250 mM EDTA, 20 mM HEPES pH 7.4). 90 uL from each reaction are transferred.
Measurement: IC50 = 0.38 nM (pChEMBL 9.420000076293945)
Descriptors: MW 578.68, ALogP 4.56, HBA 10, HBD 2, PSA 121.01, QED 0.23
Compound (SMILES): CN(C)CC[C@H](CSc1ccccc1)Nc1sc(S(=O)(=O)NC(=O)c2ccc(N3CCN(CC=C(c4ccccc4)c4ccccc4)CC3)cc2)cc1[N+](=O)[O-]
InChIKey: QLOVRKDYNJCKSX-PGUFJCEWSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 μM (2× starting concentration; 10% DMSO) and 10 μL were transferred into a 384-well plate. Then 10 μL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki = 51.7 nM (pChEMBL 7.289999961853027)
Descriptors: MW 811.07, ALogP 7.55, HBA 11, HBD 2, PSA 128.13, QED 0.06
Compound (SMILES): COc1ccccc1NC(=O)N1CCN(Cc2cnc3ccccc3c2)CC1
InChIKey: IFOFJTXHUVSDGB-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: Biological Assay: Assay Method 1: T84 frozen pellets (contents of 1-4x15 cm culture dishes) were homogenized in 300 mL of FAAH assay buffer (125 mM Tris, 1 mM EDTA, 0.2% glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH 9). The assay mixture was prepared from 50 uL of the cell homogenate, 10 uL of the test compound, and 40 uL of anandamide [1-3H-ethanolamine] (3H-AEA; Perkin-Elmer, 10.3 Ci/mmol), which was added last, for a final tracer concentration of 200 nM. The reaction mixture was incubated at rt for 1 hour (h). During the incubation, 96-well Multiscreen filter plates (catalog number MAFCNOB50; Millipore, Bedford, Mass., USA) were loaded with 25 uL of activated charcoal (Multiscreen column loader, catalog number MACL09625, Millipore) and washed once with 100 uL of MeOH. Also during the incubation, 96-well DYNEX MicroLite plates (catalog number NL510410) were loaded with 100 uL of MicroScint40 (catalog number 6013641, Packard Bioscience, Meriden, Conn., USA).
Measurement: IC50 = 540 nM (pChEMBL 6.269999980926514)
Descriptors: MW 376.46, ALogP 3.59, HBA 4, HBD 1, PSA 57.7, QED 0.76
Compound (SMILES): CC(c1ccc(Cl)cc1)c1ccc(-c2nc3cc(C(N)=O)ccc3[nH]2)cc1
InChIKey: VSASOXHZSMSDCC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Chk2 (SINGLE PROTEIN), Homo sapiens
UniProt: O96017
Assay: Inhibition of recombinant human CDS1 using N-terminal biotin-labeled SGLYRSPSMPENLNRPR as substrate in presence of [gamma-33P]-adenosine triphosphate after 3 hrs by scintillation counting method
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 375.86, ALogP 5.13, HBA 2, HBD 2, PSA 71.77, QED 0.52
Compound (SMILES): O=C(OC(C(F)(F)F)C(F)(F)F)N1CCN(Cc2ccc(Br)cc2Oc2ccccc2)CC1
InChIKey: YMORRVPLNYWPEQ-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Mus musculus
UniProt: O08914
Assay: In Vitro Competitive Activity-Based Protein Assay: Proteomes (mouse brain membrane fraction or cell lysates) (50 uL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37 C. After 30 min, FP-Rh (1.0 uL, 50 uM in DMSO) was added and the mixture was incubated for another 30 min at 37 C. Reactions were quenched with SDS loading buffer (50 uL-4x) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL, ABHD6 and FAAH using ImageJ 1.43u software.Preparation of Mouse Brain Proteomes from Inhibitor Treated Mice. Inhibitors were administered to wild-type C57Bl/6J by oral gavage in a vehicle of polyethylene glycol. Each animal was sacrificed 4 h following administration and brain proteomes were prepared and analyzed according to previously established methods (See Niphakis, M. J., et al. (2011) ACS Chem. Neurosci. and Long, J. Z., et al. Nat. Chem. Biol. 5:37-44).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 541.29, ALogP 5.99, HBA 4, HBD 0, PSA 42.01, QED 0.44
Compound (SMILES): CC(C)Oc1cccc(-c2c[nH]c3ccc(-c4nc(N)no4)cc23)n1
InChIKey: UWODNALMAWQXCS-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 = 42.74 nM (pChEMBL 7.369999885559082)
Descriptors: MW 335.37, ALogP 3.65, HBA 6, HBD 2, PSA 102.85, QED 0.59
Compound (SMILES): O=C(O)CCCCNC(=O)c1ncc2cc(-c3ccc(F)cc3)ccc2c1O
InChIKey: CXCKWUPISMEDBQ-UHFFFAOYSA-N
Target: Prolyl hydroxylase EGLN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KS0
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 18480 nM (pChEMBL 4.730000019073486)
Descriptors: MW 382.39, ALogP 3.73, HBA 4, HBD 3, PSA 99.52, QED 0.54
Compound (SMILES): Cc1ccc(-c2ccc(C(=O)N[C@@H](Cc3ccccc3)C(N)=O)cn2)s1
InChIKey: IMZIQYFZJGRQAG-KRWDZBQOSA-N
Target: Hematopoietic prostaglandin D synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O60760
Assay: Biochemical Assay: The H-PGDS catalyzed conjugation of GSH and 1-chloro-2,4-dinitrobenzene (CDNB) was used as the biochemical assay for enzyme inhibition. Reactions were performed in 96 well plate format, and product formation was followed at A340 nm over a 10 min interval at 25° C. using a POWERWAVE XS microplate scanning spectrophotometer (Bio-Tek Instruments, Winooski, Vt., USA). Reactions were performed in 0.1 M Tris HCl, pH 8.0 containing 2 mM MgCl2, 1 mM CDNB, 2 mM GSH, 2.5 ng/μ1 purified H-PGDS and 10% (v/v) ethanol in a 200 μl reaction volume. IC50 values were calculated from rates of conjugation activity determined at eight concentration points bracketing the IC50, where compound solubility allowed, and were corrected for background activity at the same solvent concentrations. All compounds were made up in 100% DMSO and diluted with 0.1 M Tris HCl, pH 8.0 with 2 mM MgCl2.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 365.46, ALogP 2.94, HBA 4, HBD 2, PSA 85.08, QED 0.7
Compound (SMILES): CC[C@H](C)[C@H](NC(=O)C[C@H](N)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)CC(C)C)C(C)C)C(=O)NCc1ccccc1
InChIKey: VLQVFFVXSAYFSH-OYSYKYNKSA-N
Target: Cathepsin D (SINGLE PROTEIN), Homo sapiens
UniProt: P07339
Assay: In-Vitro Fluorescence Assay: In order to identify modulators of cathepsin D activity, a continuous enzymatic test was carried out with a synthetic peptide which carries a fluorescent group (MCA=(7-methoxycoumarin-4-yl)acetyl) which is quenched by energy transfer from a Dpn (2,4 dinitrophenyl) group on the same molecule, in Greiner 384-well nb microtitre plates. Cleavage of the peptidic substrate by cathepsin D causes an increase in the fluorescence intensity. In order to determine the efficacy of substances, the time-dependent increase in the fluorescence intensity in the presence of the substance was compared with the time-dependent increase in fluorescence in the absence of substances. The reference substance used was pepstatin A (Sigma-Aldrich). The substrate used was MCAGKPILFFRLK(Dnp)d-R-NH2 (Enzo Life Sciences, Lorrach). The enzyme employed was cathepsin D isolated from the human liver (Sigma-Aldrich) in a final concentration of 1.4 nM. The test was carried out in 100 mM sodium acetate buffer, 1.25% (v/v) of DMSO, 0.25% (w/v) of Chaps, pH 5.5. 2 ul of each substance solution with serially diluted substance concentration were added to in each case 4 ul of cathepsin D solution and incubated at room temperature for 10 min. The reaction was started by addition of 2 ul of substrate solution (final concentration 5 uM). After carrying out a starting-point fluorescence measurement (excitation wavelength 340 nm/emission wavelength 450 nm) using an Envision multilabel reader (Perkin Elmer), the reaction was incubated at room temperature for 60 min. The amount of peptide fragment cleaved off during the reaction time was subsequently measured by determination of the increase in the fluorescence intensity at 450 nm (excitation wavelength 340 nm).
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 706.97, ALogP 4, HBA 6, HBD 6, PSA 171.52, QED 0.11
Compound (SMILES): O=C1CCc2c(Oc3ccc4c(c3)C3C(O4)C3c3nc4ccncc4[nH]3)ccnc2N1
InChIKey: QTUSAODLDZOJAR-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf IC50 Assay: Compounds disclosed herein were tested against B-Raf (V600E) (PV3849, from Invitrogen) or C-Raf (Y340D/Y341D) (PV3805, from Invitrogen) in a time-resolved fluorescence energy transfer assay. The assay was carried out in reactions (10 uL) containing 0.0625 nM B-Raf or 0.5 nM C-Raf, 25 mM Tris pH7.4, 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3BO4, 5 mM beta-glycerophosphate, 0.01% Triton X-100, 2.5 mM DTT, 0.1% BSA, 0.1 mM ATP, 13.7 nM GST-tagged MEK1 (Full-length protein with K97R mutation, recombinant protein purified from bacterial expression system) and 0-5 uM compounds disclosed herein (final concentration of 1% DMSO). The enzyme was incubated with the compounds at room temperature for 60 minutes and the reactions were initiated by the addition of ATP and GST-MEK1. After incubating at room temperature for 60 minutes, an equal volume of stop buffer containing 25 mM Tris pH7.4, 400 mM KF, 50 mM EDTA, 0.01% BSA, 0.01% Triton X-100, 1 test of Eu3+ Cryptate-conjugated rabbit polyclonal antibody anti-Phospho MEK1/2 (Ser217/221) and 1 test of d2-conjugated mouse monoclonal antibody anti-glutathione S-transferase was added to stop the reactions. Plates were sealed and incubated at room temperature for 2 hours, and then the TR-FRET signals were read on BMG PHERAstar FS instrument. The IC50 for each compound was calculated by non linear regression by Graphpad Prism software.
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 411.42, ALogP 3.67, HBA 6, HBD 2, PSA 102.02, QED 0.53
Compound (SMILES): CCCN1CCC(c2ccc(Nc3nccc(-c4c(-c5ccc(OC)c(C(=O)Nc6c(F)cccc6F)c5)nc5ccccn45)n3)c(OC)c2)CC1
InChIKey: HVTCTUCTHJXITI-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human GST-rTEV-tagged IGF1R (957 to 1367 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 703.79, ALogP 8.34, HBA 9, HBD 2, PSA 105.91, QED 0.14
Compound (SMILES): C[C@H]1COCCN1c1nnc(-c2ccc3[nH]cc(-c4cncc(C5CC5)n4)c3c2)o1
InChIKey: UMFRMBNWQDGSCQ-ZDUSSCGKSA-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 = 13.86 nM (pChEMBL 7.860000133514404)
Descriptors: MW 402.46, ALogP 3.78, HBA 7, HBD 1, PSA 92.96, QED 0.56
Compound (SMILES): CSCCCN1CCC(CC#N)(n2cc(C(N)=O)c(Nc3ccccc3)n2)CC1
InChIKey: MPFFYCBPHHYDMY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 412.56, ALogP 3.18, HBA 7, HBD 2, PSA 99.97, QED 0.61
Compound (SMILES): Cc1cc(C)n(CC(C)NC(=O)c2ccc(NC(=O)N3Cc4cccnc4C3)cc2)n1
InChIKey: BTMCTEOIUHGDHF-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 162 nM (pChEMBL 6.789999961853027)
Descriptors: MW 418.5, ALogP 3.26, HBA 5, HBD 2, PSA 92.15, QED 0.67
Compound (SMILES): Cc1noc(C)c1Cn1cc(N2C(=O)N(CCc3cc(F)cc(F)c3)C(C)(C)C2=O)cn1
InChIKey: VADXGTADQOYHOY-UHFFFAOYSA-N
Target: Taste receptor type 2 member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYW2
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 400 nM (pChEMBL 6.400000095367432)
Descriptors: MW 443.45, ALogP 3.6, HBA 6, HBD 0, PSA 84.47, QED 0.54
Compound (SMILES): C[C@H]1C[C@@H](N)CN(c2ccncc2Nc2ncc3ccc(-c4ccccc4F)nn23)C1
InChIKey: OTWLCRXTCIMBPE-JKSUJKDBSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM 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.
Measurement: IC50 < 0.1 nM (pChEMBL 10)
Descriptors: MW 417.49, ALogP 3.85, HBA 7, HBD 2, PSA 84.37, QED 0.52
Compound (SMILES): O=C(NC1COCC1O)c1ccc2cc(Oc3ccc(C(F)(F)F)cn3)ccc2n1
InChIKey: BZBSDLNDXXIWMI-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 1460 nM (pChEMBL 5.840000152587891)
Descriptors: MW 419.36, ALogP 2.93, HBA 6, HBD 2, PSA 93.57, QED 0.68
Compound (SMILES): COC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccc(NS(=O)(=O)O)cc1)c1nc2c(s1)CCCC2
InChIKey: BSDMZFVYAMVOBN-VXKWHMMOSA-N
Target: Receptor-type tyrosine-protein phosphatase beta (SINGLE PROTEIN), Homo sapiens
UniProt: P23467
Assay: Inhibition of recombinant HPTPbeta (unknown origin) preincubated for 10 mins followed by substrate addition measured after 15 mins by DiFMUP probe-based fluorescence assay
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 558.68, ALogP 3.6, HBA 7, HBD 4, PSA 146.72, QED 0.28
Compound (SMILES): Cc1c(N(Cc2ccc(F)c(C(F)(F)F)c2)S(=O)(=O)c2ccc(C3=NOS(=O)N3)cc2)sc2ccccc12
InChIKey: CRXQVBLRAONTFB-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 7.2 nM (pChEMBL 8.140000343322754)
Descriptors: MW 583.61, ALogP 5.62, HBA 6, HBD 1, PSA 88.07, QED 0.3
Compound (SMILES): C[C@@H](c1nc2ccc(N3CCC[C@@H](C(=O)N4CCCC4)C3)nc2[nH]1)n1cc(F)cn1
InChIKey: MXZDUQBFEKBHFI-LSDHHAIUSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: In Vitro DGAT2 Assay: For determination of IC50 values, the reactions were carried out in 384-well white Polyplates (Perkin Elmer) in a total volume of 20 uL. To 1 uL of compounds dissolved in 100% DMSO and spotted at the bottom of each well, 5 uL of 0.04% bovine serum albumin (BSA) (fatty acid free, Sigma Aldrich) was added and the mixture was incubated at room temperature for 20 minutes. To this mixture, 10 uL of hDGAT2 membrane fraction (0.01 mg/mL) diluted in 100 mM Hepes-NaOH, pH 7.4, 20 mM MgCl2 containing 200 nM methyl arachidonyl fluorophosphonate (Cayman Chemical; dried from ethyl acetate stock solution under argon gas and dissolved in DMSO as 5 mM stock) was added. After this mixture was preincubated at room temperature for 2 hours, DGAT2 reactions were initiated by the addition of 4 uL of substrates containing 30 uM [1-14C]decanoyl-CoA (custom-synthesized by Perkin Elmer, 50 mCi/mmol) and 125 uM 1,2-didecanoyl-sn-glycerol (Avanti Polar Lipids) dissolved in 12.5% acetone. The reaction mixtures were incubated at room temperature for 40 min and the reactions were stopped by addition of 5 uL of 1% H3PO4. After the addition of 45 uL MicroScint-E (Perkin-Elmer), plates were sealed with Top Seal-A covers (Perkin-Elmer) and phase partitioning of substrates and products was achieved using a HT-91100 microplate orbital shaker (Big Bear Automation, Santa Clara, Calif.). Plates were centrifuged at 2,000×g for 1 min in an Allegra 6R Centrifuge (Beckman Coulter) and then were sealed again with fresh covers before reading in a 1450 Microbeta Wallac Trilux Scintillation Counter (Perkin Elmer). DGAT2 activity was measured by quantifying the generated product [14C]tridecanoylglycerol in the upper organic phase.
Measurement: IC50 = 88.6 nM (pChEMBL 7.050000190734863)
Descriptors: MW 411.49, ALogP 2.74, HBA 6, HBD 1, PSA 82.94, QED 0.71
Compound (SMILES): O=C(CN1CCN(c2ccnc3cc(Cl)ccc23)CC1)Nc1cc(C(F)(F)F)ccc1Cl
InChIKey: XTNZTEVRNLRTBZ-UHFFFAOYSA-N
Target: DNA gyrase subunit B (SINGLE PROTEIN), Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
UniProt: A0QNE0
Assay: Inhibition of Mycobacterium smegmatis 155 6His-tagged DNA gyrase B catalytic domain ATPase activity expressed in Escherichia coli BL21 (DE3) pLysS cells assessed as inorganic phosphate release after 120 mins in presence of ATP by malachite green dye based assay
Measurement: IC50 = 31300 nM (pChEMBL 4.5)
Descriptors: MW 483.32, ALogP 5.32, HBA 4, HBD 1, PSA 48.47, QED 0.55
Compound (SMILES): COc1cc(OC)cc(N(CCN2CCOC2=O)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: MBVVTXSJBVWMFV-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 489.78 nM (pChEMBL 6.309999942779541)
Descriptors: MW 474.52, ALogP 3.64, HBA 9, HBD 0, PSA 94.84, QED 0.38
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1cnn(Cc2ncoc2-c2ccccc2)c1
InChIKey: SLKNIQINJJTWTN-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In-vitro Plasma Kallikrein Inhibition: Enzymatic reactions were conducted in â¿¿assay bufferâ¿¿, comprising 50 mM Hepes/NaOH at pH 7.8, 150 mM NaCl, 1 mM EDTA and 0.05% (w/v) CHAPS. For the determination of IC50 values, the assays were performed at room temperature in 384-well plates with a total assay volume of 25.25 ul per well. The test compound was dissolved in 90% (v/v) DMSO/water. For the assays, 250 nL of the 90% (v/v) DMSO/water solution or compound solution were added per well, followed by the addition of 12.5 ul protease solution (protease in assay buffer). The final assay concentration of the human plasma kallikrein was nominally 25 pM, the 11 compound concentrations in the dilution series were in the range form 1 nM to 100 uM. After 1 hour of pre-incubation at room temperature, the reactions were started by the addition of 12.5 ul substrate solution (in assay buffer, final assay concentration was 0.5 uM). After the addition of the substrate solution, the final DMSO concentration in the assay was 0.9% (v/v).
Measurement: IC50 = 3900 nM (pChEMBL 5.409999847412109)
Descriptors: MW 402.46, ALogP 3.11, HBA 7, HBD 2, PSA 111.86, QED 0.51
Compound (SMILES): O=C(NCc1ccc(S(=O)(=O)c2cccc(OC(F)(F)F)c2)cn1)c1cnc2[nH]ncc2c1
InChIKey: LQUDNHDFESTRSO-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT Enzymatic Reaction. The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mixture was incubated for 15 min at rt, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 pM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 min at rt, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 53.7 nM (pChEMBL 7.269999980926514)
Descriptors: MW 477.42, ALogP 3.01, HBA 7, HBD 2, PSA 126.93, QED 0.44
Compound (SMILES): O=C(OCC12CCN(CC1)CC2)c1c[nH]c2ccc(O)cc12
InChIKey: OJJZFBYHLRFVDB-UHFFFAOYSA-N
Target: Neuronal acetylcholine receptor subunit alpha-7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q05941
Assay: Radioligand Binding Assay: Protocol 1: The assay was conducted following the literature reference Meyer E. M., et al., Analysis of 3-(4-Hydroxy, 2-Methoxybenzylidene) Anabaseine Selectivity and Activity at Human and Rat Alpha-7 Nicotinic Receptors, J. Pharmacol. Exp. Ther., 287: 918-925, 1998. The assay was conducted using the following materials: receptor source: rat brain, radioligand: [125I] α-Bungarotoxin (2200 Ci/mmol) with a final concentration of 1 nM, non-specific determinant: Methyllycaconitine (MLA) [1 μM], reference compound: Methyllycaconitine (MLA), and positive control: Methyllycaconitine (MLA). Incubation Conditions: Reactions were carried out in 20 mM HEPES (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2 and 1.2 mM MgSO4 at 37° C. for 90 minutes. The reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined by liquid scintillation counting and compared to control values in order to ascertain any interactions of test compound with the nicotinic, ganglionic binding site.
Measurement: Ki = 132 nM (pChEMBL 6.880000114440918)
Descriptors: MW 300.36, ALogP 2.52, HBA 4, HBD 2, PSA 65.56, QED 0.85
Compound (SMILES): Cn1c(=O)n(CC(C)(C)C)c2ccc(C3=CC4CCC3CN4C(=O)Cn3cncn3)nc21
InChIKey: WNILPHCEVJNPJL-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Fluorescence Laser Imaging Plate Reader (FLIPR) Based Assay: The compounds of the present invention may be tested in a fluorescence laser imaging plate reader (FLIPR) based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr-cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with dose responses of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity. The potentiation response is monitored after a subsequent addition of an EC20 concentration of glutamate (900 nM).
Measurement: EC50 = 56.06 nM (pChEMBL 7.25)
Descriptors: MW 435.53, ALogP 2.08, HBA 8, HBD 0, PSA 90.84, QED 0.63