lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN(CCNC(=O)c1ccc(F)c(NC(=O)c2cnc3cc(-c4ccnn4C)ccn23)c1)C(C)(C)C
InChIKey: PDSOSXMAHFIZIR-UHFFFAOYSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q05030
Assay: Inhibition of PDGFR-beta driven proliferation of rat A10 cells after 68 hrs in presence of rat recombinant PDGF-BB by cell titer-glo luminescence assay
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 491.57, ALogP 3.59, HBA 7, HBD 2, PSA 96.56, QED 0.41
Compound (SMILES): CC(C)Oc1ccc(-c2nc(-c3cccc4c3CC[C@H]4NCC(=O)NCCO)no2)cc1C#N
InChIKey: STUIFOBOGFZIAO-OAQYLSRUSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Agonist activity at human S1P1 expressed in CRE-bla CHOK1 cells assessed as inhibition of forskolin-induced cAMP formation preincubated for 4 hrs followed by FRET-based beta-lactamase fluorescent substrate addition measured after 2 hrs
Measurement: EC50 = 0.05 nM (pChEMBL 10.300000190734863)
Descriptors: MW 461.52, ALogP 2.75, HBA 8, HBD 3, PSA 133.3, QED 0.44
Compound (SMILES): Cn1c(=O)nc2[nH]nc(/C=C/c3ccccc3)nc-2c1=O
InChIKey: UMFNKVICJQZYQM-BQYQJAHWSA-N
Target: Dihydrolipoyl dehydrogenase (SINGLE PROTEIN), Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
UniProt: P9WHH9
Assay: Inhibition Assay: The assay was performed in a manner similar to that described in Bryk et al., Biochemistry (2010) 49:1616-1627 and modified for an online robotics screening system.A solution containing Lpd (100 nM) and 100 mM sodium phosphate buffer (pH 7.0) was prepared. Another solution containing the substrate lipoamide (2 mM), 100 mM sodium phosphate buffer (pH 7.0), EDTA (4 mM), and NADH (200 uM) was also prepared. The detection reagent, DTNB was dissolved in DMSO and diluted to a concentration of 375 uM with a 100 mM sodium phosphate buffer (pH 7.0).4 uL/well of Lpd solution was dispensed into 1536-well clear bottom plates. Next, 50 nL of each test compounds (1 mM dissolved in DMSO) was added into each well. After one hour of incubation, 4 uL/well of lipoamide (LPA) solution was added, so that in each well, the concentrations of Lpd, LPA, and the test compound were 50 nM, 1 mM, and 6.25 uM, respectively.
Measurement: IC50 = 2160 nM (pChEMBL 5.670000076293945)
Descriptors: MW 281.28, ALogP 0.53, HBA 6, HBD 1, PSA 93.53, QED 0.74
Compound (SMILES): CC(C)Oc1ccc(-c2nc(-c3cccc4c3CC[C@@H]4NCc3cncs3)no2)cc1C#N
InChIKey: DALBQDVGZBDVIO-QFIPXVFZSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Agonist activity at human S1P1 expressed in CRE-bla CHOK1 cells assessed as inhibition of forskolin-induced cAMP formation preincubated for 4 hrs followed by FRET-based beta-lactamase fluorescent substrate addition measured after 2 hrs
Measurement: EC50 = 0.05 nM (pChEMBL 10.300000190734863)
Descriptors: MW 457.56, ALogP 5.3, HBA 8, HBD 1, PSA 96.86, QED 0.4
Compound (SMILES): CN1CCC(NC(=O)c2cccc(Nc3nccc(-c4cnc(N5CC[C@H](O)C5)c(C#N)c4)n3)c2)CC1
InChIKey: ORORMSCBPGPSRN-QHCPKHFHSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 498.59, ALogP 2.55, HBA 9, HBD 3, PSA 130.3, QED 0.47
Compound (SMILES): NS(=O)(=O)c1ccc(NC(=O)c2ccc(N3C4=C(C(=O)CCC4)C(c4cccc(-c5nnn[nH]5)c4)C4=C3CCCC4=O)cc2)cc1
InChIKey: XFGFNNUMJQYGIQ-UHFFFAOYSA-N
Target: Carbonic anhydrase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P00918
Assay: Inhibition of human recombinant carbonic anhydrase 2 expressed in Escherichia coli assessed as reduction in CO2 hydration preincubated for 15 mins prior to testing measured for 10 to 100 secs by stopped-flow assay
Measurement: Ki = 49 nM (pChEMBL 7.309999942779541)
Descriptors: MW 635.71, ALogP 4.38, HBA 9, HBD 3, PSA 181.1, QED 0.28
Compound (SMILES): Cc1cc(C)c(CN2CCc3ccc(O[C@@H]4CCOC4)c(Cl)c3C2=O)c(=O)[nH]1
InChIKey: GCKHKRQQHZWJPK-OAHLLOKOSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition of wild-type EZH2 (unknown origin) expressed in baculovirus infected SF9 cells co-expressing SUZ12/EED/RbAp48 complex using HeLa cells derived oligonucleosomes as substrate after 60 mins in presence of [3H] SAM by scintillation counting
Measurement: IC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 402.88, ALogP 3.01, HBA 4, HBD 1, PSA 71.63, QED 0.85
Compound (SMILES): C[C@]12CC[C@H]3[C@@H](CC(O)C45CC4CC[C@]35C)C1=CC=C2c1cccnc1
InChIKey: QIZMKKCGLBWKJQ-BCYWUAHMSA-N
Target: Steroid 17-alpha-hydroxylase/17,20 lyase (SINGLE PROTEIN), Homo sapiens
UniProt: P05093
Assay: Inhibition of CYP17 in human testicular microsomes using 17alpha-hydroxypregnenolone as substrate assessed as decrease in DHEA levels after 90 mins by ELISA
Measurement: IC50 <= 11 nM (pChEMBL 7.959000110626221)
Descriptors: MW 347.5, ALogP 5.01, HBA 2, HBD 1, PSA 33.12, QED 0.77
Compound (SMILES): Fc1ccc2c(c1)N1CCNCC1CC2
InChIKey: QLQOXYNICXAHQG-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2C (SINGLE PROTEIN), Homo sapiens
UniProt: P28335
Assay: Agonist activity at unedited 5-HT2C receptor (unknown origin) expressed in HEK293 cells assessed as [3H]inositol phosphate accumulation after 2 hrs by scintillation counting
Measurement: EC50 = 0.7943 nM (pChEMBL 9.100000381469727)
Descriptors: MW 206.26, ALogP 1.55, HBA 2, HBD 1, PSA 15.27, QED 0.69
Compound (SMILES): CCN1CCN(Cc2ccc(-c3cc4c(-c5cccc(N6CCOc7cc(C8CC8)cc(F)c7C6=O)c5CO)ccnc4[nH]3)nc2)CC1
InChIKey: HCGIQPXUFZJBIW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition of recombinant human full length BTK preincubated for 10 mins followed by Tk-biotin peptide substrate addition after 90 mins by TR-FRET assay
Measurement: IC50 = 2.6 nM (pChEMBL 8.59000015258789)
Descriptors: MW 646.77, ALogP 5.98, HBA 7, HBD 2, PSA 97.82, QED 0.21
Compound (SMILES): C[C@H]1CNC(=O)c2cc3ccc(C(=O)Nc4cccnc4)cc3n2[C@H]1C
InChIKey: DUPMGAVJLVBVOW-STQMWFEESA-N
Target: Ribosomal protein S6 kinase alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15349
Assay: Kinase Glo Plus Assay: Human RSK2 protein, purchased from Invitrogen, is used to measure kinase activity utilizing Kinase Glo Plus (Promega) a homogeneous assay technology, which uses a luciferin-luciferase based ATP detection reagent to quantify residual ATP. The assay is performed using 0.75 nM His-RSK2, 0.75 .mu.M ATP and 1.0 uM S6 Kinase/RSK Substrate Peptide 1 (Upstate, catalog #12-124), in assay buffer consisting of 25 mM HEPES, pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 50 mM KCl, 0.2% BSA, 0.01% CHAPS, 100 uM Na3VO4, 0.5 mM DTT, and 1% DMSO. Solutions of test compounds at various concentrations are prepared by 1:3 fold serial dilution of a 1 mM solution of compound in DMSO. The DMSO solutions are further diluted with assay buffer to a final concentration of DMSO of 5%. The assay is performed in a 384 well, white, non-binding plate (Corning, catalogue #3574). Solutions of test compounds (10 uL) are transferred to a dry assay plate, followed by addition of 20 uL kinase.
Measurement: IC50 = 240 nM (pChEMBL 6.619999885559082)
Descriptors: MW 348.41, ALogP 3.23, HBA 4, HBD 2, PSA 76.02, QED 0.75
Compound (SMILES): CNC(=O)c1cccc(Nc2ncnc(N3CCC(c4ccc(C#N)cc4)CC3)n2)c1
InChIKey: INCFVQCBRFMTDM-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Cell-Based Assay: Compounds were tested on human Nav1.7 (hNav1.7) expressed stably in 293 cells, which have no endogenous sodium channels. Two forms of whole-cell patch-clamp assays were used. In both, peak current through Nav1.7 was evoked by a depolarizing voltage pulse, and currents were monitored as a function of time in response to exposure to several different concentrations of drug. Automated electrophysiology uses the PatchXpress 7000A system. Up to sixteen cells at a time were voltage-clamped in parallel, and the steady-state voltage dependence of inactivation was determined experimentally for each cell. For each cell, fractional inactivation was set at 20%, and peak current through Nav1.7 as a function of time was measured in response to increasing concentrations of test compound. Manual electrophysiology refers to conventional whole-cell patch-clamping (Hamill et al., 1981). After formation of the whole-cell clamp, cells were lifted off the bottom of the dish with the patch pipette and positioned directly in front of an array of glass microperfusion tubes each with internal diameter about 250 microns, with individual compound concentrations flowing from each tube. Current as a function of time in response to increasing drug concentrations was measured with Nav1.7 fully in the resting state (holding voltage set to −140 mV) and on the same cells with the holding voltage set to produce about 20% inactivation. Compounds were not applied for a prespecified time, but for as long as required to achieve a stable level of block.
Measurement: IC50 = 4300 nM (pChEMBL 5.369999885559082)
Descriptors: MW 413.49, ALogP 3.23, HBA 7, HBD 2, PSA 106.83, QED 0.66
Compound (SMILES): C[C@H]1C[C@H]2CSC(N)=N[C@@]2(c2nc(NC(=O)c3ccc(OC(F)F)cc3)cs2)CO1
InChIKey: OPDKSXUPVRRQFC-DKHZXNSXSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 Cell-Free Assay: The substrate is Biotin-GLTNIKTEEISEISY^EVEFR-C[Oregon Green]KK-OH. The BACE1 enzyme is affinity purified material from conditioned media of CHO-K1 cells that have been transfected with a soluble BACE construct (BACE1deltaTM96His). Compounds are incubated in a ½ log dose response curve from a top concentration of 100 μM with BACE1 enzyme and the biotinylated fluorescent peptide in 384-well black plates (Thermo Scientific #4318). BACE1 is at a final concentration of 0.1 nM with a final concentration of peptide substrate of 150 nM in a reaction volume of 30 μL assay buffer [100 mM sodium acetate, pH 4.5 (brought to pH with acetic acid), and 0.001% Tween-20]. Plates are covered and incubated for 3 hours at 37 °C. The reaction is stopped with the addition of 30 μL of 1.5 μM Streptavidin (Pierce, #21125). After a 10 minute incubation at room temperature, plates are read on a PerkinElmer EnVision for fluorescence polarization (Ex485 nm/Em530 nm).
Measurement: IC50 = 106 nM (pChEMBL 6.96999979019165)
Descriptors: MW 454.52, ALogP 3.68, HBA 8, HBD 2, PSA 98.83, QED 0.72
Compound (SMILES): Cc1c(-c2cccc(Cl)c2)nnc(N2CCN(c3ccc(C(F)(F)F)cn3)CC2)c1C
InChIKey: IYBAQGPEVIIAGL-UHFFFAOYSA-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 447.89, ALogP 5.15, HBA 5, HBD 0, PSA 45.15, QED 0.56
Compound (SMILES): COC[C@H](O)Cc1ccc(NC(=O)c2ncc(C#N)[nH]2)c(C2=CCC(C)(C)CC2)c1
InChIKey: BDBDLANNRKYVKT-GOSISDBHSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Mus musculus
UniProt: P09581
Assay: Inhibition of c-fms in mouse bone marrow macrophages assessed as reduction in recombinant mouse CSF-1-driven cell proliferation by measuring bromodeoxyuridine incorporation incubated for 30 hrs by ELISA
Measurement: IC50 = 33.88 nM (pChEMBL 7.46999979019165)
Descriptors: MW 408.5, ALogP 3.68, HBA 5, HBD 3, PSA 111.03, QED 0.65
Compound (SMILES): Cc1noc(C)c1Cn1cc(N2C(=O)N(CCO)C(C)(C)C2=O)cn1
InChIKey: JELASXOYLZSFFB-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 = 560 nM (pChEMBL 6.25)
Descriptors: MW 347.38, ALogP 1.08, HBA 7, HBD 1, PSA 104.7, QED 0.81
Compound (SMILES): COc1cc(N)c(Cl)cc1C(=O)NCC1CCN(CCCn2ccc3ccccc32)CC1
InChIKey: BACJEQJZMAJMGA-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13639
Assay: Binding Assay: The binding affinity of the compounds for a human 5-HT4 receptor was assayed according to the method as disclosed in the literature [Wyngaert et al., Journal of Neurochemistry, (1997) 69, 1810-1819]. For this purpose, COS-7 cells expressing the human 5-HT4 receptor were constructed and homogenized to obtain membrane homogenates which were then used in binding assay experiments. For the binding assay, the membrane homogenates were respectively mixed and incubated with different concentrations of test materials and [H3]-GR113808 (Amersham Biosciences). The concentrations of the individual test materials were set to 4 uM, 1 uM, 0.25 uM, and 0.0625 uM, respectively, and the concentration of [H3]-GR113808 was set to 0.595 nM. After the incubation was completed, the reaction products were collected in GF/B glass fiber filters using a Packard cell harvester, and the bound radioactivity was then determined using a liquid cell scintillation counter (Packard TopCount NXT).
Measurement: IC50 = 0.039 nM (pChEMBL 10.40999984741211)
Descriptors: MW 455, ALogP 4.42, HBA 5, HBD 2, PSA 72.52, QED 0.49
Compound (SMILES): CC#C[C@]1(O)CC[C@H]2[C@@H]3CCc4cc(C(=O)Nc5cccnc5)ccc4[C@H]3[C@@H](O)C[C@@]21C
InChIKey: JVWNOGCJXBHUQZ-YNHSGCSHSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Antagonist activity at GR in human HepG2 cells assessed as inhibition of protein mediated-transcriptional activity by MMTV-promoter driven luciferase reporter gene assay
Measurement: IC50 = 455 nM (pChEMBL 6.340000152587891)
Descriptors: MW 430.55, ALogP 3.92, HBA 4, HBD 3, PSA 82.45, QED 0.63
Compound (SMILES): N#CC1(NC(=O)[C@@H]2Cc3ccc(c(Cl)c3)OCCCCOc3cc(Cl)ccc3C(=O)N2)CC1
InChIKey: IJYNGHYQWAZCAH-IBGZPJMESA-N
Target: Procathepsin L (SINGLE PROTEIN), Homo sapiens
UniProt: P07711
Assay: Cathepsin Enzyme Inhibition Assay: Enzyme activity is measured by observing the increase in fluorescence intensity caused by cleavage of a peptide substrate containing a fluorophore whose emission is quenched in the intact peptide. Assay buffer: 100 mM potassium phosphate pH 6.5, EDTA-Na 5 mM, Triton X-100 0.001%, DDT 5 mM. Enzymes (all at 1 nM): human and mouse Cathepsin S, Cat K, Cat B, Cat LSubstrate (20 uM): Z-Val-Val-Arg-AMC, except for Cat K which uses Z-Leu-Arg-AMC (both from Bachem). Z=Benzyloxycarbonyl. AMC=7-Amino-4-Methyl-Coumarin. Final volume: 100 uL. Excitation 360 nm, Emission 465 nm. Enzyme is added to the substance dilutions in 96-well microtitre plates and the reaction is started with substrate. Fluorescence emission is measured over 20 minutes, during which time a linear increase is observed in the absence of inhibitor.
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 488.37, ALogP 4.06, HBA 5, HBD 2, PSA 100.45, QED 0.66
Compound (SMILES): O=C(c1cc(Cl)ccc1-n1nccn1)N1CCOC[C@H]1Cc1cc(-c2ncon2)ccc1F
InChIKey: GOPNUSXTFNCXKO-QGZVFWFLSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: OX Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 ug/mL G418, 100 U/mL penicillin, 100 ug/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37 C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 468.88, ALogP 3.19, HBA 8, HBD 0, PSA 99.17, QED 0.44
Compound (SMILES): Fc1ccc(-c2n[nH]c3ccc(CCc4ccccn4)cc23)cc1
InChIKey: QEYHOTJANLQECO-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P45984
Assay: Inhibition of His6-tagged JNK2 (unknown origin) assessed as reduction in c-Jun phosphorylation using GST-c-Jun (1 to 79 residues) and [gamma32P]ATP incubated for 1 hr by scintillation counting assay
Measurement: IC50 <= 10000 nM (pChEMBL 5)
Descriptors: MW 317.37, ALogP 4.55, HBA 2, HBD 1, PSA 41.57, QED 0.6
Compound (SMILES): O=C(/C=C/c1ccc(Cl)cc1)NC[C@@H]1CCN(Cc2cc(Cl)cc(Cl)c2)C(=O)[C@H](CCN2CCCCC2)N1
InChIKey: HYVIXZUCJRMWQX-SORORRETSA-N
Target: Melanocortin receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P33032
Assay: Radioligand Binding Assay: Assessments of compound binding to human MC5R (hMC5R)) by displacement of an 125I-labeled NDP-MSH receptor ligand peptide were performed essentially as described in the data sheets produced by Perkin Elmer to accompany their frozen hMC5R membranes (Perkin Elmer catalog number RBXMC5M400UA). [125I] NDP-MSH: Radiolabeled in House and Purified by HPLC: Na125I (0.5 mCi, 17.4 Ci/mg) was added to 50 μL sodium phosphate (50 mM, pH 7.4) in an eppendorf tube precoated with IODOGEN. After incubation for 10 mins the phosphate buffer containing the iodine was added to NDP-MSH (10 μl at 1 mg/mL) in a separate eppendorf tube. This was incubated for a further 10 mins. The iodinated NDP-MSH was purified by HPLC on a Zorbax SB 300 column using solvent A: 0.05% TFA and solvent B: 90% acetonitrile 0.045% TFA with a linear gradient, 0-67% B over 60 mins. The 125I NDP-MSH eluted at 52 mins after the unlabeled starting material (48 min) and was counted and stored in the freezer. It was used within 48 hrs, as radioactive decay and ligand decomposition resulted in greatly reduced specific binding observed after 72 hrs. Incubation buffer: 25 mM HEPES-KOH (pH 7.0), 1.5 mM CaCl2, 1 mM MgSO4, 0.1 M NaCl, 1 mM 1,10-phenanthroline, and 1 Complete™ protease inhibitor tablet/100 mL (Roche, catalog number 1873580). Perkin Elmer frozen hMC5 membranes: catalog number RBXMC5M400UA, 0.4 mL/vial; 400 microassays/vial, 0.78 mg/mL protein concentration. Vials of frozen membranes were thawed rapidly immediately before use, diluted with binding buffer and vortexed. Resuspended membranes were kept on ice until they were added to the wells of the plate. Assays were performed in 96 well polypropylene plates. Membranes (0.78 μg 40 μL of a 1:40 dilution in incubation buffer) were added to [125I] NDP-MSH (0.84 nM; 2200 Ci/mmol) and test compounds in a total volume of 140 μL. This was incubated for 1 hr at 37° C. Non-specific binding was determined with 3 mM NDP-MSH. Plates were filtered using a Tomtec cell harvester with GF/A filters (Wallac) (presoaked in 0.6% polyethylenimine) and washed three times with 1.0 mL ice-cold wash buffer (the above incubation buffer without 1,10-phenanthroline and Complete™ protease inhibitor tablet). The filters were dried in a 37° C. oven, placed in a sample bag and 5 mL Betaplatescint (Wallac) was added. Prepared filters were counted in cassettes in a Microbeta Trilux (Wallac) for 1 min.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 577.98, ALogP 5.41, HBA 4, HBD 2, PSA 64.68, QED 0.39
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1COc2ccccc2N(Cc2cccc3c2ccn3-c2ccccc2C#N)C1=O
InChIKey: XVSPJSXQUDWMJL-CVDCTZTESA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: Ten nanomolar of 6x Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(8-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-C1, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 > 54800 nM (pChEMBL 4.261000156402588)
Descriptors: MW 493.57, ALogP 3.52, HBA 6, HBD 2, PSA 99.39, QED 0.43
Compound (SMILES): CCCCCCN(c1sc2ccccc2c1Br)S(=O)(=O)c1ccc(C(=O)O)cc1
InChIKey: SIJUUZTYHXHWQL-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 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 496.45, ALogP 6.14, HBA 4, HBD 1, PSA 74.68, QED 0.36
Compound (SMILES): CC#CC(=O)NCCOc1cccc(Nc2cc(-c3cc(F)cc(-n4ncc5c6c(sc5c4=O)CCCC6)c3CO)cn(C)c2=O)n1
InChIKey: XPQAHVKNSSZOHK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Biochemical Btk Assay: A generalized procedure for a standard biochemical Btk, Kinase Assay that can be used to test Formula I compounds is as follows. A master mix minus Btk enzyme is prepared containing 1x Cell Signaling kinase buffer (25 mM Tris-HCl, pH 7.5, 5 mM beta-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM MgCl2), 0.5 uM Promega PTK Biotinylated peptide substrate 2, and 0.01% BSA. A master mix plus Btk enzyme is prepared containing 1x Cell Signaling kinase buffer, 0.5 uM PTK Biotinylated peptide substrate 2, 0.01% BSA, and 100 ng/well (0.06 mU/well) Btk enzyme. Btk enzyme is prepared as follows: full length human wildtype Btk (accession number NM-000061) with a C-terminal V5 and 6xHis tag was subcloned into pFastBac vector (Invitrogen/Life Technologies) for making baculovirus carrying this epitope-tagged Btk.
Measurement: Ki = 0.306 nM (pChEMBL 9.510000228881836)
Descriptors: MW 654.72, ALogP 3.98, HBA 11, HBD 3, PSA 140.37, QED 0.16
Compound (SMILES): N#Cc1cccnc1N1CCN(c2nc(NC3CC3)n3ncc(/C=C4\NC(=O)NC4=O)c3n2)CC1
InChIKey: BAQHEOSECNOPCO-YBEGLDIGSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 471.49, ALogP 0.47, HBA 11, HBD 3, PSA 156.47, QED 0.35
Compound (SMILES): NC(=O)c1nnc(N[C@@H]2CCCC[C@@H]2N)nc1Nc1ccc2nn(C(F)F)cc2c1
InChIKey: XYFCYFFJZRCQOI-WCQYABFASA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Syk Kinase Inhibition Assay: The in vitro inhibitory activity of the compound of the present invention against Syk kinase activity was assayed under the following conditions: the purified human Syk protein used in the test were purchased from Carna Biosciences, Inc. For the inhibitory activity assay on the compound, the compound of the present invention was first serially diluted with dimethyl sulfoxide (DMSO). Next, the purified human Syk protein FL-Peptide 22 (final concentration: 1 uM) (Caliper Life Sciences, Inc.), magnesium chloride (final concentration: 5 mM), ATP (final concentration: 30 uM), and each DMSO solution of the compound of the present invention (final concentration of DMSO: 5%) were added into a buffer solution for reaction (20 mM tris-HCl pH 7.5, 20 mM NaCl, 2.5 mM DTT, 0.02% Tween-20, 1% glycerol, 0.01 mM Pefabloc), and the mixture was then incubated at 25° C. for 30 minutes to perform kinase reaction. The kinase reaction was stopped by the addition thereto of EDTA.
Measurement: IC50 = 0.52 nM (pChEMBL 9.279999732971191)
Descriptors: MW 417.42, ALogP 2.14, HBA 9, HBD 4, PSA 149.66, QED 0.48
Compound (SMILES): Cc1ccc(OCCNc2nc(Nc3cc(C)[nH]n3)cc(C)c2C#N)c(C)n1
InChIKey: KMUQNRCFLITLEM-UHFFFAOYSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Inhibition of recombinant Aurora A kinase derived from human Hela cells using kemptide as substrate in presence of [c-32P]ATP
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 377.45, ALogP 3.54, HBA 7, HBD 3, PSA 111.54, QED 0.54
Compound (SMILES): O=C(N1C[C@@H](OCC2CC2)CC[C@@H]1Cc1ccccc1)n1ncc(C(O)(c2ccc(F)cc2)c2ccc(F)cc2)n1
InChIKey: LGBQVORQOWSIEF-WDYNHAJCSA-N
Target: Diacylglycerol lipase-alpha (SINGLE PROTEIN), Mus musculus
UniProt: Q6WQJ1
Assay: Inhibition of MB064 probe binding to DAGLalpha in mouse brain membrane proteome preincubated for 30 mins followed by MB064 probe addition measured after 20 mins by gel-based competitive activity based protein profiling assay
Measurement: IC50 = 79.43 nM (pChEMBL 7.099999904632568)
Descriptors: MW 558.63, ALogP 5.31, HBA 6, HBD 1, PSA 80.48, QED 0.32
Compound (SMILES): COc1ccc(S(=O)(=O)N(Cc2ccc(F)c(C(F)(F)F)c2)c2sc3ccccc3c2Cl)cc1
InChIKey: MOELNIZXDIWMHB-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 = 84 nM (pChEMBL 7.079999923706055)
Descriptors: MW 529.96, ALogP 7.12, HBA 4, HBD 0, PSA 46.61, QED 0.25
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CCOC[C@H]2Cc2cccc(-c3ccncc3)c2)c1
InChIKey: KQPLEQHOKPZPHW-HSZRJFAPSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: OX Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 ug/mL G418, 100 U/mL penicillin, 100 ug/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37 C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES.
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 439.52, ALogP 3.72, HBA 6, HBD 0, PSA 73.14, QED 0.47
Compound (SMILES): CCCC[C@H](NC(C)=O)C(=O)N[C@H]1CCC(=O)NCC[C@@H](C(N)=O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccc(Cl)cc2)NC(=O)[C@H](CCCN)NC1=O
InChIKey: SZBAHFVIZJHMFU-VEIVPZTASA-N
Target: Melanocortin receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P32245
Assay: Competitive Inhibition Binding Assay: A competitive inhibition binding assay was performed using membrane homogenates prepared from HEK-293 cells that express recombinant hMCR-1a or hMCR-4 (in each instance where the h prefix refers to human), or alternatively membrane homogenates from B16-F10 mouse melanoma cells containing endogenous murine MCR-1. In the examples that follow, all MCR-1 and MCR-4 values are for human recombinant receptors, unless otherwise noted. Assays were performed in 96 well polypropylene round-bottom plates (VWR catalog number 12777-030). Membrane homogenates were incubated with 0.1 nM [I125]-NDP-α-MSH (Perkin Elmer) and increasing concentrations of test peptides of the present invention in buffer containing 25 mM HEPES buffer (pH 7.5) with 100 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.3 mM 1,10-phenanthroline, and 0.2% bovine serum albumin. After incubation for 90 minutes at 37° C., the assay mixture was filtered onto GF/B Unifilter plates (Perkin-Elmer catalog number 6005177) and washed with 3 mL of ice-cold buffer per well. Filters were air dried and 35 uL of scintillation cocktail added to each well. Plates were counted in a Microbeta counter for bound radioactivity. Non-specific binding was measured by inhibition of binding of [I125]-NDP-α-MSH in the presence of 1 uM NDP-α-MSH. Maximal specific binding (100%) was defined as the difference in radioactivity (cpm) bound to cell membranes in the absence and presence of 1 uM NDP-α-MSH. Each assay was conducted in duplicate and the actual mean values are described, with results less than 0% reported as 0%. Ki values for peptides of the present invention were determined using Graph-Pad Prism curve-fitting software.
Measurement: Ki = 1235 nM (pChEMBL 5.909999847412109)
Descriptors: MW 835.41, ALogP -2.21, HBA 10, HBD 12, PSA 334.71, QED 0.05
Compound (SMILES): Cc1ccc(F)c(C(=O)Nc2ccc(C#Cc3ccc4nc(N)cn4n3)cc2)c1
InChIKey: XVHXHKROMDBDGP-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: PDGFRbeta Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 ug/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 uL reaction volumes containing 2.7 uM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 385.4, ALogP 3.41, HBA 5, HBD 2, PSA 85.31, QED 0.52
Compound (SMILES): Clc1ccc2c(N3CCN(Cc4c[nH]c5ccccc45)CC3)ccnc2c1
InChIKey: YSZQCJMJIIWGKU-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 = 8300 nM (pChEMBL 5.079999923706055)
Descriptors: MW 376.89, ALogP 4.69, HBA 3, HBD 1, PSA 35.16, QED 0.56
Compound (SMILES): Cc1cc(C(=O)N[C@H]2CC[C@H](Oc3ccnc4c(Br)cccc34)CC2)no1
InChIKey: REQDIIXPFUIHKZ-HDJSIYSDSA-N
Target: Androgen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P10275
Assay: Transactivation Assay: For determining the androgen receptor-dependent transcription, a cellular assay system was used, consisting of PC-3 cells (Kaighn et al., Invest. Urol. 17: 16-23, 1979), which express the human androgen receptor stably and recombinantly (full length, wild-type form, see Swiss-Prot Acc. No. P10275, Entry Version 159, Sequence Version 2). In addition, these PC3 cells contain a stably integrated reporter gene plasmid, which is based on the commercially available plasmid pGL4.14 (#E6691, Promega Corporation, Madison, Wis., USA) and contains the luciferase gene from the American firefly (Photinus pyralis) under the control of the MMTV promoter (Cato et al., EMBO J. 6: 363-368, 1987). These cells were propagated in routine cell culture at 37° C. and 5% CO2 in a medium containing 90% RPMI 1640 (Invitrogen GmbH, Darmstadt, Germany), 100 U penicillin, 100 μg/ml streptomycin (Invitrogen), 4 mM L-glutamine (Invitrogen), 10% fetal calf serum (FCS Serum Gold, PAA Laboratories GmbH, Cölbe, Germany), 600 μg/ml Geneticin (G418-sulphate, Invitrogen) and 10 μg/ml puromycin (Sigma Aldrich GmbH, Germany). For carrying out the transactivation assays, approx. 1000 cells per well were plated out in a 384-well cell culture plate in a medium that contained activated charcoal-treated calf serum (FCS Serum Gold, PAA Laboratories) at a concentration of 5% (v/v). The test substances were added in a concentration series from 5.12×10^−12 to 1×10^−5 M in the presence of 1×10^−1° R1881 (methyltrienolone). The test plates were incubated overnight at 37° C. and 5% CO2. After 16 hours, 15 μl of Steady Glo Lysis and Detection reagent (E2550, Promega Corporation, Madison, Wis., USA) was added per well and the luminescence was read in a Topcount Luminometer (PerkinElmer, Waltham, Mass., USA) for 4 seconds per well. The luminescence values obtained were normalized, wherein 100% corresponded to the effect of the unstimulated control (without R1881), and 0% corresponded to the effect of the stimulated control (R1881 plus DMSO instead of test substance).
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 430.3, ALogP 4.41, HBA 5, HBD 1, PSA 77.25, QED 0.66
Compound (SMILES): N#Cc1ccc(N2CCN(C(=O)c3ccc(NC(=O)N4Cc5ccccc5C4)cc3)CC2)cc1
InChIKey: GONMYMUQRGEXPO-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1251667.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 13.8 nM (pChEMBL 7.860000133514404)
Descriptors: MW 451.53, ALogP 4.07, HBA 4, HBD 1, PSA 79.68, QED 0.65
Compound (SMILES): CC1(C)[C@@H]2CC[C@@]1(C)[C@@H](NC(=O)[C@H](CC1CCCCC1)NS(=O)(=O)N[C@@H](CCCNC(=N)N)C(=O)O)C2
InChIKey: IXONGSRYYIUNOM-PDGNJTGUSA-N
Target: Carboxypeptidase B2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96IY4
Assay: Inhibition of activated human plasma TAFI incubated for 15 mins by chromogenic assay
Measurement: IC50 = 49 nM (pChEMBL 7.309999942779541)
Descriptors: MW 542.75, ALogP 1.8, HBA 5, HBD 7, PSA 186.5, QED 0.1
Compound (SMILES): CCC[C@H]1CN(C(=O)C[C@@H](N)c2ccc(C(F)(F)F)cc2)CCN1c1nc(N)c2cc(OC)c(OC)c(F)c2n1
InChIKey: NKMJXIURYOBQJW-PKOBYXMFSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: ELISA Assay: CVF-Bb complex prepared from purified cobra venom factor (1 μM), recombinant human complement factor B (expressed in drosophila cells and purified using standard methods) and human complement factor D (expressed in E. Coli, refolded and purified using standard methods). CVF-Bb complex at 3 nM concentration was incubated with test compound at various concentrations for 1 hour at room temperature in PBS pH 7.4 containing 10 mM MgCl2 and 0.05% (w/v) CHAPS. Human complement C3 substrate purified from plasma was added to a final concentration of 1 μM. After 1 hour incubation at room temperature, the enzyme reaction was stopped by addition of a cocktail of concentrated pan-protease inhibitors. The product of the reaction, C3a, was quantified by means of an enzyme-linked-immunosorbent assay.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 564.58, ALogP 4.29, HBA 8, HBD 2, PSA 119.83, QED 0.39
Compound (SMILES): CCOc1ccc(-c2nc3ccccn3c2-c2ccnc(Nc3cc(CC)c(N4CCC(CCS(C)(=O)=O)CC4)cc3OC)n2)cc1C(=O)Nc1c(F)cccc1F
InChIKey: CZYNNIWHFOHBBU-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 809.94, ALogP 8.35, HBA 11, HBD 2, PSA 140.05, QED 0.11
Compound (SMILES): Cc1c(Oc2ccccc2OCCn2ccc(=O)[nH]c2=O)cc(F)c2ccc(C#N)cc12
InChIKey: OIPOKGOOHCBAPR-UHFFFAOYSA-N
Target: Human immunodeficiency virus type 1 reverse transcriptase (SINGLE PROTEIN), Human immunodeficiency virus 1
UniProt: Q72547
Assay: Inhibition of HIV1 reverse transcriptase K103N/Y181C double mutant infected in human MT2 cells assessed as protection against viral infection by MTT assay
Measurement: EC50 = 890 nM (pChEMBL 6.050000190734863)
Descriptors: MW 431.42, ALogP 3.88, HBA 6, HBD 1, PSA 97.11, QED 0.5
Compound (SMILES): Cc1nnc(Cc2nnc(N3CCN(c4ccc(C(F)(F)F)cn4)CC3)c(C)c2C)o1
InChIKey: UMMPRFMUGCIHSG-UHFFFAOYSA-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 433.44, ALogP 3.12, HBA 8, HBD 0, PSA 84.07, QED 0.62
Compound (SMILES): CCCCn1nc(C(C)(C)C)s/c1=N/C(=O)c1cc(C(F)(F)F)ccc1N/N=C\c1cccc(C)n1
InChIKey: IWDFRWZGNACPSR-WKSXEYSBSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Radioligand Binding Assay: HEK293 cells stably expressing human CB2 receptors were grown until a confluent monolayer was formed. Briefly, the cells were harvested and homogenized in TE buffer (50 mM Tris-HCl, 1 mM MgCl2, and 1 mM EDTA) using a polytron for 2x 10 second bursts in the presence of protease inhibitors, followed by centrifugation at 45,000xg for 20 minutes. The final membrane pellet was re-homogenized in storage buffer (50 mM Tris-HCl, 1 mM MgCl2, and 1 mM EDTA and 10% sucrose) and frozen at -78 C. until used. Saturation binding reactions were initiated by the addition of membrane preparation (protein concentration of 5 ug/well for human CB2) into wells of a deep well plate containing [3H]CP 55,940 (120 Ci/mmol, a nonselective CB agonist commercially available from Tocris) in assay buffer (50 mM Tris, 2.5 mM EDTA, 5 mM MgCl2, and 0.5 mg/mL fatty acid free BSA, pH 7.4). After 90 min incubation at 30 C., binding reaction was terminated by the addition of 300 uL/well of cold assay buffer.
Measurement: Ki = 185 nM (pChEMBL 6.730000019073486)
Descriptors: MW 518.61, ALogP 5.95, HBA 7, HBD 1, PSA 84.53, QED 0.31
Compound (SMILES): Cc1c(NC(=O)c2ccc(C(C)(C)C)cc2)cc(F)cc1-c1ncnc2[nH]c(C3=CC[S+]([O-])CC3)cc12
InChIKey: CWCDRMMXNVSKMN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Biochemical Enzyme Assay: The inhibitory activity of the present compounds against Btk was assessed in a biochemical enzyme assay. Assay plates in 384 well format were prepared with 8-point serial dilutions for the test compounds on a Thermo CatX workstation equipped with a Innovadyne Nanodrop Express. The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 ul per well of peptide/ATP-solution (4 uM FITC-Ahx-TSELKKVVALYDYMPMNAND-NH2, 164 uM ATP) in kinase buffer (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 18 mM MgCl2, 1 mM MnCl2) and 4.5 ul per well of enzyme solution (6.4 nM full-length human recombinant BTK) in kinase buffer. Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 ul per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Kinase reactions were analyzed on a Caliper LC3000 workstation by separating phosphorylated and unphosphorylated peptides and kinase activities were calculated from the amounts of newly formed phospho-peptide. Inhibition data were calculated by comparison to control reactions without enzyme (100% inhibition) and without inhibitors (0% inhibition).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 516.64, ALogP 6.16, HBA 4, HBD 2, PSA 93.73, QED 0.32
Compound (SMILES): COCc1nc2c(C(=O)Nc3cccc(Cl)c3C)cc(NC(=O)c3c(F)cccc3Cl)cc2[nH]1
InChIKey: WVISYJGEGOQQAX-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition Assay: mPGES-1 microsome fractions were prepared from CHO-K1 cells transiently transfected with plasmid encoding the human mPGES-1cDNA. Microsomes were diluted with potassium phosphate buffer containing reduced glutathione (pH7.4), and DMSO containing test compound or DMSO alone was added (such that DMSO final concentration would be 1% in each) and incubated at 4° C. for 20 minutes. Then, the enzymatic reactions were initiated by the addition of PGH2 substrate (final concentration 1 μM) and incubated at 4° C. for 60 seconds. The reaction was terminated by the addition of a citrate solution (final citrate concentration 50 mM) containing ferric chloride (final concentration 1 mg/mL). PGE2 production in the enzyme reaction aliquot was measured using HTRF kit (Cisbio International, catalogue #62P2APEC).
Measurement: IC50 = 6.7 nM (pChEMBL 8.170000076293945)
Descriptors: MW 501.35, ALogP 5.97, HBA 4, HBD 3, PSA 96.11, QED 0.3
Compound (SMILES): COC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3c3cc(Cl)c(Cl)cc3F)ccc2[nH]1
InChIKey: DUOZVKLOQOJVHC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 501.3, ALogP 5.04, HBA 5, HBD 3, PSA 107.55, QED 0.34
Compound (SMILES): CCN(CC)CCN(CC12CC(c3ccc(C(F)(F)F)cc3)(C1)C2)C(=O)Cn1c(SCc2ccc(F)cc2)nc(=O)c2c1CCC2
InChIKey: DPBVEZITSOYBHD-UHFFFAOYSA-N
Target: Platelet-activating factor acetylhydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: Q13093
Assay: Inhibition of human recombinant LpPLA2
Measurement: IC50 = 0.3981 nM (pChEMBL 9.399999618530273)
Descriptors: MW 656.79, ALogP 6.47, HBA 6, HBD 0, PSA 58.44, QED 0.12