lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(c1cccc(NS(=O)(=O)N2CCCC2)c1F)c1c[nH]c2ncc(-c3ccccc3)cc12
InChIKey: NCHWSWXNTPPTOV-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Biochemical activity: Assays for biochemical and cell based activity are known in the art, for example, as described in PCT publication WO 2007/002433, the disclosure of which is hereby incorporated by reference as it relates to such assays. For example, the biochemical activity IC50 values are determined with respect to inhibition of B-Raf V600E kinase activity or p-Erk kinase activity, where inhibition of phosphorylation of a peptide substrate is measured as a function of compound concentration. Compounds to be tested are diluted in dimethyl sulfoxide to a concentration of 0.1 mM. These are serially diluted 15 uL into 30 uL of dimethyl sulfoxide seven times in 96 well plates for a total of 8 dilution points, and for each dilution point 1 uL is added to a well of an assay plate. Plates are prepared such that each well in a 384 well plate contains 1 uL of compound in 10 uL volume with 0.1 ng Raf enzyme (i.e. any of B-Raf, c-Raf-1 or B-Raf V600E, Upstate Biotechnology or prepared by methods known to one of skill in the art), 50 mM HEPES, pH 7.0, 50 mM NaCl, 2 mM MgCl2, 1 mM MnCl2, 0.01% Tween-20, 1 mM DTT, and 100 nM biotin-MEK1 as substrate. The reaction is started with addition of 10 uL of 200 uM ATP (i.e. final 100 uM ATP). After incubation of the kinase reaction for 45 minutes at room temperature, 5 uL/well of Stop Solution is added (25 mM Hepes pH 7.5, 100 mM EDTA, 0.01% BSA with donor beads (Streptavidin coated beads, Perkin Elmer), acceptor beads (Protein A coated, Perkin Elmer), and anti phosphor MEK1/2 antibody (CellSignal), each at final concentration 10 ug/mL).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 464.52, ALogP 4.35, HBA 4, HBD 2, PSA 95.16, QED 0.41
Compound (SMILES): COc1cccc2c1CCN(C(=O)[C@H]1CC(c3cccc(Cl)c3F)=NO1)[C@H]2C(=O)Nc1ccc(C(=O)O)cc1
InChIKey: JMRJXBGKNVHKIB-ILBGXUMGSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Enzyme Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 73.9 nM (pChEMBL 7.130000114440918)
Descriptors: MW 551.96, ALogP 4.44, HBA 6, HBD 2, PSA 117.53, QED 0.47
Compound (SMILES): O=C(O)C1COc2cc(COc3ccc(-c4ccn[nH]4)nc3)ccc2C1
InChIKey: UYIFWRKPLBWPHG-UHFFFAOYSA-N
Target: Cytochrome P450 4F2 (SINGLE PROTEIN), Homo sapiens
UniProt: P78329
Assay: Inhibition Test for Each Compound of the Present Invention Against 20-HETE Producing Enzymes (CYP4F2 and CYP4A11): In the CYP4F2 inhibition test, the reaction solution containing each compound [final concentration of 50 mM, KPO4 (pH 7.4), 2.5 μM luciferine derivative, and 1 mM NADPH] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4F2 had been expressed. In the CYP4A11 inhibition test, the reaction solution containing each compound [final concentration of 100 mM, Tris-HCl (pH 7.5), 60 μM luciferine derivative, 1.3 mM NADP+, 3.3 mM Glucose 6-Phosphate, 3.3 mM MgCl2, and 0.4 U/mL Glucose 6-Phosphate dehydrogenase] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4A11 had been expressed. Following this, the membrane fraction was left to stand at room temperature for 60 minutes to perform an enzymatic reaction. After the reaction, a luciferine detection reagent was added, and the luminescence value was measured using a plate reader. By using that value, the percent inhibition of 20-HETE producing enzyme (%) was calculated according to the equation described below, and the 50% inhibitory concentration (IC50 value) for each compound was calculated.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 351.36, ALogP 2.69, HBA 5, HBD 2, PSA 97.33, QED 0.73
Compound (SMILES): CCOC(=O)Cn1cc(Nc2ncc3ccn(CC4CCCCCC4)c3n2)cn1
InChIKey: XGSCTQZBMIARPA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK/TYK2 Assay: 10 mM test compound stock or 1 mM control compound stock (tofocitinib, ruxolitinib or staurosporine) in DMSO was diluted to 0.4 mM in DMSO. A 3-fold series dilution was then performed in DMSO to generate 10 different compound concentrations. The assay was carried out in 384-well white plate. 0.5 uL of 40× compound DMSO solution at different concentrations was mixed with 10 uL 2× enzyme prepared in reaction buffer (20 mM HEPES, 10 mM MgCl2, 0.01% Tween, 1 mM DTT, pH 7.5). 10 uL 2× substrate mixture prepared in reaction buffer was then added to start the reaction. A short spin was done to settle down all solutions to the bottom of the plate. Final concentrations of test compound in the reaction mixture were 10000, 3333, 1111, 370, 123, 41.2, 13.7, 4.57, 1.52and 0.51 nM. Concentrations of control compound were ten times less. Enzymatic reaction was conducted at 25° C. for 1-2 hours. 10 uL of Kinase Glo Reagents was added to stop the reaction and generate the luminescent signal which was measured using Envision. Luminescence signal was inversely related to kinase activity. Reaction mixture which did not contain enzyme served as negative control. The mixture without any compound was the positive control.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 396.5, ALogP 3.9, HBA 8, HBD 1, PSA 86.86, QED 0.48
Compound (SMILES): CCOc1cc2ncc(C#N)c(Nc3ccc(NC(=O)c4ncccc4C)c(Cl)c3)c2cc1NC(=O)CCCN(C)C
InChIKey: ZSBOVIKNCKVUHX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13043
Assay: MST1/MST2 Biochemical LanthaScreen Eu Kinase Binding Assay: MST1 and MST2 biochemical LanthaScreen Eu Kinase Binding Assay was based on the binding and displacement of kinase tracer to the kinase of interest. Compounds in 1000×DMSO stock solution was dispensed using automated dispensing system (Labcyte) to 384 well Corning Microplate at 15 nL, 5 uL of Kinase buffer A was added to each well. Plates were shaken and incubated for 1 min to ensure well dissolution of compounds. Kinase/Antibody mixture was added at a final concentration of 5 nM and 2 nM and kinase tracer 222 solution at a final concentration of 100 nM in a total volume of 20 uL. Plates were incubated for 1.5 hrs in dark at room temperature and assay plates were scanned on Envision plate reader with excitation: 340 nM and kinase Tracer Emission at 665 nM.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 586.1, ALogP 6.14, HBA 8, HBD 3, PSA 132.27, QED 0.19
Compound (SMILES): NCCC(=O)N[C@H]1CC[C@H](N(CCO)C(=O)c2cc(C3CC3)on2)CC1
InChIKey: USUGJKIVWYAQHQ-HDJSIYSDSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), Tris, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), and Tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP) were purchased from Sigma-Aldrich at the highest level of purity possible. 3H-SAM was purchase from American Radiolabeled Chemicals with a specific activity of 80 Ci/mmol. 384-well opaque white OptiPlates and SPA beads (Perkin Elmer, catalog # RPNQ0013) were purchased from PerkinElmer.
Measurement: IC50 = 16790 nM (pChEMBL 4.78000020980835)
Descriptors: MW 364.45, ALogP 0.76, HBA 6, HBD 3, PSA 121.69, QED 0.63
Compound (SMILES): CN1Cc2ccc(NC(=O)c3n[nH]c4cc(F)c(-c5cnn(C)c5)cc34)cc2C1
InChIKey: AKQJAOKBKBKHTP-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: Kinase Assay: DYRK1A: Each compound was dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 11-point dose-response curves from 10 M to 0.00016 M) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 1536-well black-walled round bottom plates (Corning).The DYRK1A kinase assay was run using the Ser/Thr 18 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologiesa Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as a ratio of coumarin emission/fluorescein emission.Briefly, recombinant DYRK1A kinase, ATP and Ser/Thr peptide 18 were prepared in 1× Kinase buffer to final concentrations of 0.19 μg/mL, 30 μM, and 4 μM respectively. The mixture was allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 served as control reactions. Additionally, an 11-point dose-response curve of Staurosporine (1 uM top) was run to serve as a positive compound control.After incubation, Development Reagent A was diluted in Development Buffer then added to the reaction and allowed to further incubate for one hour at room temperature. The plate was read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer).
Measurement: EC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 390.42, ALogP 3.3, HBA 5, HBD 2, PSA 78.84, QED 0.56
Compound (SMILES): O=c1cc(C2CCNCC2)n2ncc(-c3nc(-c4cccc(C(F)(F)F)n4)no3)c2[nH]1
InChIKey: ZYCGCBPBCMLVJG-UHFFFAOYSA-N
Target: Tissue-type plasminogen activator (SINGLE PROTEIN), Homo sapiens
UniProt: P00750
Assay: Plasma-based Clot Lysis Assay: The clot-lysis test system configures the kinetics of clot formation and degradation in vitro and allows quantifying modulation of the process by selected test compounds.The test compounds were dissolved in 1% acetic acid and further complemented with an equal volume of DMSO. The resulting stock solutions were serially diluted in 0.5% acetic acid/50% DMSO. 1 μL aliquots of these solutions were placed into 384 well microplates (Greiner, black, transparent bottom), followed by 30 μL of diluted human citrated plasma (platelet-poor, final concentration: 5%; supplemented with fibrinogen, final concentration: 3 μM; dilution buffer: 20 mM HEPES, 150 mM NaCl, 0.01% Brij (pH 7)). The reactions were started by addition of 20 μL of CaCl2 (final concentration: 10 mM), and tPA (tissue plasminogen activator, final concentration: 0.2 nM) in dilution buffer, followed by an additional volume of 20 μL dilution buffer for improved mixing. The reactions were incubated at 37° C. Clot formation and degradation was monitored spectrophotometrically by kinetic optical density measurements at 405 nm. IC50 values were determined by comparing the resulting time courses with the time course of a blank control reaction.
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 431.38, ALogP 2.62, HBA 8, HBD 2, PSA 114, QED 0.51
Compound (SMILES): OC(c1ncc[nH]1)c1ccc(F)cc1N1CC2CC2C1
InChIKey: GHIAQYPZQHTFJE-UHFFFAOYSA-N
Target: Proteinase-activated receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P55085
Assay: IP-One HTRF Assay: Inositol monophosphate (IP1) production were measured in 1321N1 cells stably expressing human PAR2 using an IP-One HTRF assay kit (Cisbio). 80 nl of compound in 100% DMSO were added in white small-volume 384-well plates using an Echo 555 (Labcyte) and were incubate for 30 min at 37° C. with 4 μl of cells (15,000 cells per well) in stimulation buffer (HBSS with 20 mM HEPES, pH 7.4). IP1 production was initiated by addition of 4 μl of 140 μM SLIGRL-NH2 (SEQ ID NO.: 4) in stimulation buffer supplemented with 100 mM LiCl. After 60 min at 37° C. cells were lysed and IP1 concentrations were detected according to the manufacturers protocol (Cisbio). Data were normalized to IP1 concentrations using a IP1 standard curve according to the manufacturers protocol (Cisbio).
Measurement: IC50 = 4670 nM (pChEMBL 5.329999923706055)
Descriptors: MW 273.31, ALogP 2.09, HBA 3, HBD 2, PSA 52.15, QED 0.9
Compound (SMILES): Cc1c(-c2c(C)c3c(c(C)c2[C@H](OC(C)(C)C)C(=O)O)CN(C(=O)c2cc(F)c(F)c(F)c2)C3)cc(F)c2c1CCCO2
InChIKey: TZEYWZMGJAFESK-PMERELPUSA-N
Target: Human immunodeficiency virus type 1 integrase (SINGLE PROTEIN), Human immunodeficiency virus 1
UniProt: Q7ZJM1
Assay: Anti-HIV Activity MT4 Assay: Antiviral HIV activity and cytotoxicity values for compounds of the invention from Table 1 were measured in parallel in the HTLV-1 transformed cell line MT-4 based on the method previously described (Hazen et al., 2007, In vitro antiviral activity of the novel, tyrosyl-based human immunodeficiency virus (HIV) type 1 protease inhibitor brecanavir (GW640385) in combination with other antiretrovirals and against a panel of protease inhibitor-resistant HIV (Hazen et al., In vitro antiviral activity of the novel, tyrosyl-based human immunodeficiency virus (HIV) type 1 protease inhibitor brecanavir (GW640385) in combination with other antiretrovirals and against a panel of protease inhibitor-resistant HIV, Antimicrob. Agents Chemother. 2007, 51: 3147-3154; and Pauwels et al., Sensitive and rapid assay on MT-4 cells for the detection of antiviral compounds against the AIDS virus, J. of Virological Methods 1987, 16: 171-185).
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 599.62, ALogP 7.26, HBA 4, HBD 1, PSA 76.07, QED 0.25
Compound (SMILES): Cc1nn(C(F)F)cc1S(=O)(=O)N1C[C@H](CC(=O)NS(=O)(=O)C2CC2)Oc2ccc(NC(=O)OCC(C)(C)C)cc21
InChIKey: IIBQMHFLWBTXJX-INIZCTEOSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biological Assay: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., a biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.
Measurement: IC50 = 534 nM (pChEMBL 6.269999980926514)
Descriptors: MW 619.67, ALogP 3.14, HBA 10, HBD 2, PSA 166, QED 0.43
Compound (SMILES): NC/C(=C\F)COc1ccc(-c2nc(N3CCOCC3)n[nH]2)c(F)c1
InChIKey: OCWCFWIISDLZJE-DHZHZOJOSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: Enzyme Activity Assay:: The inhibition of the sample on VAP-1 enzyme activity was measured by using an Amplex® Red Monoamine Oxidase kit (Invitrogen #A12214). 100 nL of the gradiently-diluted to-be-tested compound (solvent DMSO) was added to a 384-well plate. 25 μL of 10 nM VAP-1 enzyme solution was added and incubated for 30 minutes at room temperature. A substrate mixture of VAP-1 enzyme (200 μM Amplex Red, 1 U/mL HRP, 1 mM Benzylamine) was added and incubated for 60 minutes at room temperature. After the incubation, the fluorescent signal was read with a microplate reader Envision (excitation light wavelength 530-560 nm, emission light wavelength 590 nm). The fluorescence signal value after the background signal was removed was analyzed by Prism software, and the IC50 of the sample against the VAP-1 enzyme was calculated.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 351.36, ALogP 1.64, HBA 6, HBD 2, PSA 89.29, QED 0.82
Compound (SMILES): CCOC(=O)N1CCc2cc(F)c(C(=O)Nc3cccc(-c4nnc5n4C(C)(C)CC5)n3)cc2C1
InChIKey: YICNKIWDBQXXEP-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: HTRF KinEASE Assay: ASK1 was purchased from Thermofisher (Catalogue #PV4011), ATP was purchased from Sigma (Catalogue #A7699), HTRF® KinEASE Assay System was obtained from Cisbio (Bedford, Mass.). ½ Area plate was purchased from Perkin Elmer (Catalogue ##6005560). HTRF® KinEASE-STK is a generic method for measuring serine/threonine kinase activities using a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. The IC50 value for each compound was determined in the presence of compound (various concentration from 0 to 10 μM) and a fixed amount of ATP and peptide substrates. The test compound, 1 μM STK3 peptide substrate, and 5 μM of ASK1 kinase are incubated with kinase reaction buffer containing 50 mM HEPES Ph 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1 mM EGTA for 30 minutes. 100 μM ATP is added to start kinase reaction and incubated for 3 hours. The STK3-antibody labeled with Eu3+-Cryptate and 125 nM streptavidin-XL665 are mixed in a single addition with stop reagents provided by the Cisbio kit used to stop the kinase reaction. Fluorescence is detected using an Envision Multilabeled 2014 reader from PerkinElmer. The Fluorescence is measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm is calculated for each well. The resulting TR-FRET is proportional to the phosphorylation level. Staurosporine was used as the positive control. IC50 was determined by Xlfit 5.3.
Measurement: IC50 = 5.6 nM (pChEMBL 8.25)
Descriptors: MW 478.53, ALogP 3.93, HBA 7, HBD 1, PSA 102.24, QED 0.61
Compound (SMILES): COc1cc(-c2cccc(C(F)(F)F)c2)c(C)cc1-c1ncnc2cc(S(=O)(=O)Nc3ccon3)ccc12
InChIKey: KIMZPRPQQCAIHC-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to â¿¿10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 18.4 nM (pChEMBL 7.739999771118164)
Descriptors: MW 540.52, ALogP 6.09, HBA 7, HBD 1, PSA 107.21, QED 0.28
Compound (SMILES): CO[C@@H]1[C@H](OC(=O)N2CC(CCN3CCC(F)(F)CC3)C2)CC[C@]2(CO2)[C@H]1[C@@]1(C)O[C@@H]1CC=C(C)C
InChIKey: LTJQTTILFMTEBN-AUKZVGPFSA-N
Target: Methionine aminopeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P50579
Assay: Biological Enzymatic Assay: Flag tagged Human recombinant MetAP2 expressed and isolated for use as the enzyme source. 10 mM stock solutions of compounds were prepared in 100% DMSO and further diluted in 100% DMSO required concentration to 1 mM stocks. The stock compound solutions and DMSO vehicle controls were diluted to target final compound concentrations using assay buffer to a final concentration of 50 mM HEPES containing 100 mM NaCl, pH adjusted to 7.5. The MAS peptide was formulated to a 7.5 mM stock in distilled water and prior to use further diluted 1:4. Amino acid oxidase was prepared as a stock solution (6.2 mg/ml) and prior to use further diluted 1:49.6 in distilled water. A 250 μM solution of MnCl2 was prepared in advance of thawing an aliquot of MetAP2 enzyme. 40 μl of enzyme was mixed with 100 μl of MnCl2 then further diluted in assay buffer to a final concentration of 16 μg/ml. To test for compound effect on MetAP2 enzyme activity, 5 μl of test compound, 10 μl of MAS substrate/amino acid oxidase mixture, 10 μl of MetAP2 was added to test wells in a 384 well black plate with blank wells containing no enzyme, replaced with 10 μl of assay buffer. All compounds were tested in duplicate on two occasions on the same day. The final in well concentrations of the assay were: 1% DMSO, 0.272 μg/ml MetAP2, 10 μM MnCl2, 50.0 μg/ml (0.225 U/ml) amino acid oxidase, and 0.75 mM MAS. The plate was sealed with a TopSeal A cover and mixed briefly on an orbital mixer at 900 rpm. The plate was incubated for a further 25 minutes at 25° C. A 5× stock of Amplex buffer was prepared (0.25M sodium phosphate, pH 7.4) and stored at 4° C. When preparing for use the stock was diluted with distilled water Amplex Ultraread stock solution was prepared at 2.57 mg/ml in 100% DMSO and stored in 50 μl aliquots at −20° C. 20 μl of 505 U/ml. Horse radish peroxidase was diluted in 990 ml of Amplex buffer, 100 μl of this was combined with 50 μl of Amplex Ultrared in 4850 ml of 1× Amplex buffer to generate sufficient detection reagent for a 384 well plate. 25 μl detection reagent was added to each well of the test plate, which was re-sealed and mixed briefly on an orbital shaker. The plate was transferred to an Envision Multi-label reader and RFU measured corresponding to excitation 531 nm and emission 595 nm At the end of the MetAP2 incubation 25 μl Amplex/HRP mixture per well was added and the plate read plate on a plate reader.
Measurement: IC50 = 275 nM (pChEMBL 6.559999942779541)
Descriptors: MW 512.64, ALogP 4.25, HBA 6, HBD 0, PSA 67.07, QED 0.36
Compound (SMILES): O=C1NCCN(CC[C@H]2CC[C@](O)(C(F)(F)F)CC2)c2c1oc1ccc(C(F)(F)F)cc21
InChIKey: RJGQFKGLZBIDSQ-HHCCDPJTSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Pim-1 Inhibitory Activity Assay: The calculation of the Pim-1 inhibitory activity of the compound was performed using the following solution according to the protocol attached to ADP-Glo Kinase Assay (cat. V9102, Promega). The purified enzyme of human Pim-1 described above was used. (i) Preparation of Solution. The kinase buffer solution (50 mmol/L HEPES (pH 7.5), 5 mmol/L MgCl2, 1 mmol/L DTT, 0.05% BSA) was prepared by dissolving HEPES (Jena Bioscience), MgCl2 (Sigma-Aldrich), DTT (Sigma-Aldrich) and BSA (Sigma-Aldrich) in purified water. The ATP solution (288 μmol/L) was prepared by dissolving 100 mmol/L ATP (Promega) in the kinase buffer solution. The enzyme/substrate solution (0.2 mmol/L Pim-1, 30 μmol/L Pim2tide) was prepared by dissolving Pim-1 (described above) and Pim2tide (custom synthetic product by GenScript USA, the same product as PIM2tide cat. 12-542 by Millipore) in the kinase buffer solution. The test compound solution (containing 12.5% DMSO) was prepared by dissolving the DMSO solution of the test compound in the kinase buffer solution. The vehicle solution (containing 12.5% DMSO) was prepared by dissolving DMSO in the kinase buffer solution. (ii) Method. To a 384-well assay plate (Corning, 4513), the test compound solution or vehicle solution (control) was added by 1 μL/well, the enzyme/substrate solution or kinase buffer solution (blank) was added by 2 μL/well, and the ATP solution was added by 2 μL/well, and they were mixed. After enzymatically reacting at room temperature for 45 minutes, ADP-Glo Reagent (Promega) was added by 5 μL/well, and they were mixed. After reacting at room temperature for 60 minutes, Kinase Detection Reagent (Promega) was added by 10 μL/well, and they were mixed. After reacting at room temperature for 30 minutes, the luminescence amount of each well was measured for 10 msec with a multi-label plate reader EnVision (PerkinElmer).
Measurement: Ki = 4.08 nM (pChEMBL 8.390000343322754)
Descriptors: MW 464.41, ALogP 4.88, HBA 4, HBD 2, PSA 65.71, QED 0.64
Compound (SMILES): Cc1sc2c(-c3cc(Cl)ccc3C#CCn3c(C)nc4cc(C(F)(F)F)c(N5CCN(C)CC5)c(C#N)c4c3=O)ccnc2c1C(=O)O
InChIKey: VZFAGGBTBDJRRT-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 13.1 nM (pChEMBL 7.880000114440918)
Descriptors: MW 691.14, ALogP 6.34, HBA 9, HBD 1, PSA 115.35, QED 0.22
Compound (SMILES): Cc1cnc(COCC(=O)Nc2cc(-c3[nH]c4c(c3Nc3ccccc3)C(=O)CCC4)ccn2)o1
InChIKey: ATWRZQFBQOYDSP-UHFFFAOYSA-N
Target: Mitotic checkpoint serine/threonine-protein kinase BUB1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43683
Assay: Kinase Assay: In a typical assay 11 different concentrations of each compound (0.1 nM, 0.33 nM, 1.1 nM, 3.8 nM, 13 nM, 44 nM, 0.15 μM, 0.51 μM, 1.7 μM, 5.9 μM and 20 μM) were tested in duplicate within the same microtiter plate. To this end, 100-fold concentrated compound solutions (in DMSO) were previously prepared by serial dilution (1:3.4) of 2 mM stocks in a clear low volume 384-well source microtiter plate (Greiner Bio-One, Frickenhausen, Germany), from which 50 nl of compounds were transferred into a black low volume test microtiter plate from the same supplier. Subsequently, 2 μL of Bub1 (the final concentration of Bub1 was adjusted depending on the activity of the enzyme lot in order to be within the linear dynamic range of the assay: typically 200 ng/mL were used) in aqueous assay buffer [50 mM Tris/HCl pH 7.5, 10 mM magnesium chloride (MgCl2), 200 mM potassium chloride (KCl), 1.0 mM dithiothreitol (DTT), 0.1 mM sodium ortho-vanadate, 1% (v/v) glycerol, 0.01% (w/v) bovine serum albumine (BSA), 0.005% (v/v) Trition X-100 (Sigma), 1× Complete EDTA-free protease inhibitor mixture (Roche)] were added to the compounds in the test plate and the mixture was incubated for 15 min at 22° C. to allow pre-equilibration of the putative enzyme-inhibitor complexes before the start of the kinase reaction, which was initiated by the addition of 3 μL 1.67-fold concentrated solution (in assay buffer) of adenosine-tri-phosphate (ATP, 10 μM final concentration) and peptide substrate (1 μM final concentration). The resulting mixture (5 μL final volume) was incubated at 22° C. during 60 min., and the reaction was stopped by the addition of 5 μL of an aqueous EDTA-solution (50 mM EDTA, in 100 mM HEPES pH 7.5 and 0.2% (w/v) bovine serum albumin) which also contained the TR-FRET detection reagents (0.2 μM streptavidin-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-phosho-Serine antibody [Merck Millipore, cat. #35-002] and 0.4 nM LANCE EU-W1024 labeled anti-mouse IgG antibody [Perkin-Elmer, product no. AD0077, alternatively a Terbium-cryptate-labeled anti-mouse IgG antibody from Cisbio Bioassays can be used]). The stopped reaction mixture was further incubated 1 h at 22° C. in order to allow the formation of complexes between peptides and detection reagents.
Measurement: IC50 = 6.45 nM (pChEMBL 8.1899995803833)
Descriptors: MW 471.52, ALogP 4.79, HBA 7, HBD 3, PSA 122.14, QED 0.34
Compound (SMILES): CCCCS(=O)(=O)Nc1cc(-c2cc(C)c(=O)n(C)c2)nc(Oc2c(C)cccc2C#N)n1
InChIKey: PMRXWTIFRSPRCA-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: In Vitro Enzyme Inhibition Assay-CREBBP Inhibition: Determination of the IC50 for the CREBBP inhibitors disclosed herein was performed as follows: CREBBP was cloned and expressed in E. Coli as His-tag protein and purified by Nickel affinity and gel-filtration chromatography. The protein was further characterized as a single band with the correct molecular weight by SDS-PAGE. CREBBP binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Perkin Elmer). In a 384-well ProxiPlate CREBBP (50 nM final) was combined with peptide (20 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20-minute incubation at room temperature, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After two hours of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit.The ability of the compounds disclosed herein to inhibit CBP activity was quantified and the respective IC50 value was determined.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 467.55, ALogP 3.66, HBA 8, HBD 1, PSA 126.97, QED 0.54
Compound (SMILES): CC(=O)c1cccc(-c2cnc3ccc(NCc4ccco4)nn23)c1
InChIKey: JXNFOBLGDRURHX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Fyn (SINGLE PROTEIN), Homo sapiens
UniProt: P06241
Assay: ADP-Glo Kinase Assay: The assay procedure determines the IC50 of each potential FYN kinase inhibitor by measuring the enzyme catalyzed ATP-dependent phosphorylation of the FYN substrate peptide. The ADP-Glo Kinase Assay is specifically designed to quantify kinase activity by measuring the ADP produced in the reaction The reaction buffer comprises 40 mM Tris.HCl (pH 7.5), 20 mM MgCl2, 0.1 mg/ml BSA and 5 microM DTT. Individual compounds within the chemical library were dissolved in DMSO at known concentrations. Briefly, 2.5 microliters of reaction buffer containing 3 picograms of FYN kinase and serial dilutions of each potential FYN kinase inhibitor (9 3-fold dilutions of a 10 micromolar top sample) are placed into each well of a 384 well microtiter plate and incubated at room temperature for 30 minutes. An additional 2.5 microliters of reaction buffer containing 100 picograms of the FYN substrate and 12.5 picomoles of ATP are then added to each well and the reaction carried out for 90 minutes at room temperature.
Measurement: IC50 = 997 nM (pChEMBL 6)
Descriptors: MW 332.36, ALogP 3.8, HBA 6, HBD 1, PSA 72.43, QED 0.56
Compound (SMILES): CC(O)c1nc2nc(-c3noc(=O)[nH]3)nc(N[C@H](C)C3CCC3)c2n1C[C@H]1CC[C@H](C)CC1
InChIKey: BRDBBOLRHVOOHG-QKGHYUKFSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 81.1 nM (pChEMBL 7.090000152587891)
Descriptors: MW 455.56, ALogP 3.65, HBA 9, HBD 3, PSA 134.75, QED 0.49
Compound (SMILES): COc1cc(C(=O)NS(C)(=O)=O)c(F)cc1-c1cnc(OCC2(C)CCCCC2)c(Cl)c1
InChIKey: PQXPZAQIBNOLNG-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav 1.7 In Vitro Patch Express (PX) Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to −10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes. Peak sodium current was measured at the end of the compound addition period to determine percent inhibition. Three to five cells were tested per concentration, and IC50 curves were fitted to percent inhibition as a function of concentration.
Measurement: IC50 = 98.6 nM (pChEMBL 7.010000228881836)
Descriptors: MW 484.98, ALogP 4.59, HBA 6, HBD 1, PSA 94.59, QED 0.62
Compound (SMILES): COCC1(n2c(NS(=O)(=O)[C@@H](C)[C@H](OC)c3ncc(Cl)cn3)nnc2-c2ccn(C)n2)CC1
InChIKey: NFGWYYXRTFGLPW-WFASDCNBSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPgammaS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in 9 μL assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl and 0.1% (w/v) BSA], 1 μL of diluted test compound (starting with 0.75 mM, 2-fold serial dilution with DMSO, total 22 points), 10 μL of 18 μM GDP (final concentration of 3 μM GDP), 20 μL of 0.25 μg/mL membrane protein expressing human APJ receptor captured with WGA PS beads (final concentration of 5 μg per well), and 20 μL of 0.3 nM [35S]GTPγS (final concentration is 0.1 nM [35S]GTPγS)(Perkin Elmer Life and Analytical Sciences, Waltham USA). One column of the plate was 1 μL of DMSO as background and another column of the plate was 1 μL of 180 μM Pyr-Apelin-13 which was used as control at a final concentration of 3 μM. Incubation was at RT for 90 min and the microplate was read using a ViewLux ultra HTS Microplate Imager (PerkinElmer, Inc.). All the results presented are means of several independent experiments and analyzed by non-linear regression methods using the commercially available program Prism (GraphPad, San Diego, Calif.) providing the EC50 values detailed in Table 40.
Measurement: EC50 = 9.9 nM (pChEMBL 8)
Descriptors: MW 496.98, ALogP 1.78, HBA 11, HBD 1, PSA 138.94, QED 0.44
Compound (SMILES): CN(c1cccc(C(=O)NO)c1)c1nc2cc(-c3cccnc3)c(C(F)(F)F)cc2n1C
InChIKey: OUNVUCRCBOQDIR-UHFFFAOYSA-N
Target: Histone deacetylase 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96DB2
Assay: HDAC Inhibition Assays: This example describes in vitro inhibition properties of exemplary HDAC11 inhibitors for various HDACs. HDAC inhibition assays were performed using an electrophoretic mobility shift assay at Nanosyn, Inc. (Santa Clara, Calif.). Full length human recombinant HDAC proteins were expressed in the baculoviral system and purified by affinity chromatography. A human recombinant HDAC3 was co-expressed with nuclear receptor corepressor (Ncor2). The following peptide substrates were used: FAM-RHKK(Ac)-NH2 for HDAC3, HDAC6 and HDAC8; FITC-H3K27(Ac)-NH2 for HDAC1, HDAC2 and HDAC10; FAM-RHKK(tri-fluor-Ac)-NH2 for HDAC4, HDAC5, HDAC7, HDAC9 and HDAC11. Reactions consisting of compound, enzyme, and substrate were performed in reaction buffer (comprised of 100 mM HEPES, pH7.5, 25 mM KCl, 0.1% bovine serum albumin, 0.01% Triton X-100) at 25° C. and quenched by the addition of termination buffer (100 mM HEPES, pH7.5, 0.01% Triton X-100, 0.05% SDS). The fluorescence intensity of the electrophoretically separated de-acetylated product and substrate peptide were measured and analyzed using the LabChip® 3000 microfluidic electrophoresis instrument (Perkin Elmer/Caliper Life Sciences). The IC50 values of inhibitors were determined by fitting the %-inhibition curves with 4 parameter dose-response model using XLfit 4 software (IDBS).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 441.41, ALogP 4.54, HBA 6, HBD 2, PSA 83.28, QED 0.36
Compound (SMILES): CCN(C)[C@]1(c2ccccc2)CC[C@]2(CC1)CN(CC(C)(C)C(N)=O)C(=O)N2CC1(O)CCC1
InChIKey: XEMGIQPBMSNWHD-LDNJAJMQSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Receptor Binding Assay: MOP: The hMOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl (pH 7.4) supplemented with 0.052 mg/ml bovine serum albumin (Sigma-Aldrich Co. St. Louis. Mo.). The final assay volume (250 μl/well) included 1 nM of [N-allyl-2.3-3H]naloxone as ligand (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). and either test compound in dilution series or 25 μM unlabelled naloxone for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration. which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd. Buckinghamshire. UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). After incubation for 90 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux ß-counter (PerkinElmer Life Sciences/Wallac. Turku. Finland). Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]naloxone-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation.
Measurement: Ki = 2615 nM (pChEMBL 5.579999923706055)
Descriptors: MW 470.66, ALogP 3.31, HBA 4, HBD 2, PSA 90.11, QED 0.61
Compound (SMILES): O=C1CCc2cc(/C=C/C(=O)N3CCC(c4ccccc4)CC3)cnc2N1
InChIKey: WMXWUKJBKJQVJC-RMKNXTFCSA-N
Target: Enoyl-[acyl-carrier-protein] reductase [NADPH] FabI (SINGLE PROTEIN), Staphylococcus aureus
UniProt: Q6GI75
Assay: Enzyme Inhibition Assay: FabI enzyme inhibition assays were carried out in half-area, 384-well microtitre plates. Compounds were evaluated in 40-μl assay mixtures containing 100 mM NaADA, pH 6.5 (ADA=N-[2-acetamido]-2iminodiacetic acid), 250 μM crotonoyl-CoA, 625 μM NADH and 50 μg/ml S. aureus ATCC 29213 FabI Inhibitors were typically varied over the range of 50 to 0.39 μM. The reaction mixtures were incubated for 30 minutes at room temperature and the reaction was stopped by adding 200 mM Tris buffer (pH 9.0) to create a pH-shift. The consumption of NADH was monitored by measuring the change in absorbance at 340. By comparing sample readings to those of negative (absence of compound) and positive (absence of enzyme) controls, the percent inhibition of enzymatic activity of the compounds was determined A best-fit curve is fitted by a minimum of squares method. From this an IC50-value (expressed in μg/ml), resulting in 50% inhibition of enzymatic activity, was obtained.
Measurement: IC50 = 802 nM (pChEMBL 6.099999904632568)
Descriptors: MW 361.45, ALogP 3.39, HBA 3, HBD 1, PSA 62.3, QED 0.85
Compound (SMILES): Cc1cccc(C)c1-n1nc2c(c1-c1cc(F)c(NC(N)=O)cc1F)CN(c1ccc(Cl)cc1F)CC2
InChIKey: KPFAPIQWLQJRFC-UHFFFAOYSA-N
Target: C5a anaphylatoxin chemotactic receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21730
Assay: chemotaxis assay: Optionally, compounds may be further assayed for their ability to inhibit chemotaxis in cells. Chemotaxis assays are performed using 5 μm pore polycarbonate, polyvinylpyrrolidone-coated filters in 96-well chemotaxis chambers (Neuroprobe; Gaithersburg, Md.) using chemotaxis buffer (Hank's balanced salt solution (HBSS) and 1% FBS). C5aR ligands (i.e., C5a, R&D Systems; Minneapolis, Minn.) are use to evaluate compound mediated inhibition of C5aR mediated migration. Other chemokines (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) are used as specificity controls. The lower chamber is loaded with 29 μl of chemokine (i.e., 0.03 nM C5a) and varying amounts of compound; the top chamber contains 100,000 U937 or neutrophil cells in 20 μl. The chambers are incubated 1.5 hours at 37° C., and the number of cells in the lower chamber quantified either by direct cell counts in five high powered fields per well or by the CyQuant assay (Molecular Probes), a fluorescent dye method that measures nucleic acid content and microscopic observation.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 525.96, ALogP 6.28, HBA 4, HBD 2, PSA 76.18, QED 0.33
Compound (SMILES): N#Cc1cccc(Oc2ccc(/N=c3\[nH]c(=O)n(CCC(=O)O)c(=O)n3Cc3ccc(Cl)cc3)cc2)n1
InChIKey: LVFXZTMCDMAENT-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 518.92, ALogP 2.81, HBA 9, HBD 2, PSA 155.36, QED 0.36
Compound (SMILES): NCCCC[C@H](NC(=O)C1CCCC1)C(=O)COc1c(F)c(F)cc(F)c1F
InChIKey: DRRLOKQBVIVNIT-AWEZNQCLSA-N
Target: Lys-gingipain (SINGLE PROTEIN), Porphyromonas gingivalis (strain ATCC 33277 / DSM 20709 / JCM 12257)
UniProt: B2RLK2
Assay: Inhibition Activity Assay: The capacities of compounds of the present invention to inhibit the activity of lysine gingipain (Kgp) were measured in a fluorogenic assay similar to those described by Barret (Biochemical Journal. 1980, 187(3), 909) using 10 M Z-His-Glu-Lys-MCA as a fluorogenic substrate at 37Â° C. By testing a range of concentrations for each compound, the concentration required to inhibit the activity of lysine gingipain by 500(the âIC50â) was determined.
Measurement: IC50 < 0.05 nM (pChEMBL 10.300999641418457)
Descriptors: MW 404.4, ALogP 2.99, HBA 4, HBD 2, PSA 81.42, QED 0.36
Compound (SMILES): CNc1cc2c(cn1)c(-c1cnn(C)c1)nn2C1CCC(Oc2cccnn2)CC1
InChIKey: HKBYHKNYHRHQLA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Enzyme Assay: AXL: Compounds of Formula I were screened for their ability to inhibit AXL kinase using Invitrogen's LanthaScreen Eu Kinase Binding technology. His-tagged recombinant human AXL cytoplasmic domain was incubated with 20 nM Alexa-Fluor® Tracer 236 (PR9078A), 2 nM biotinylated anti-His (Cat. No. M4408), and 2 nM europium-labeled Streptavidin (Cat. No. PV5899) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using a concentration of control compound that completely inhibits the enzyme. The POC values are fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.
Measurement: IC50 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 404.48, ALogP 3.23, HBA 9, HBD 1, PSA 95.57, QED 0.55
Compound (SMILES): CCN1C(=O)c2c(O)c(=O)c(C(=O)NCc3cccc(Cl)c3F)cn2C[C@]12C[C@@H](COC)C2
InChIKey: ZDUYCWBPNKNUJX-WXHQJSEUSA-N
Target: Protein Nef (SINGLE PROTEIN), Human immunodeficiency virus 1
UniProt: P03406
Assay: Measurement of Antiviral (HIV-1) Activity: The medium (40 μL), a test substance (10 μL) diluted with the medium, and a 1×105 cells/mL MT-4 cell suspension (50 μL) wherein HIV-1 NL4-3 strain was infected with MOI (infection multiplicity) 0.05 were added to each well of a 96-well black plate (manufactured by Corning Incorporated), and the mixture was cultured at 37° C. for 5 days.medium composition: RPMI1640, 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin.Then, Cell Titer-Glo (manufactured by Promega Corporation, 100 μL) was added to each well, and the mixture was stood at room temperature for 10 min, and the luminescence intensity was measured.
Measurement: EC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 477.92, ALogP 2.55, HBA 6, HBD 2, PSA 100.87, QED 0.67