lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(Nc1ccc(OC(F)(F)F)cc1)c1cnc(OCCNCCO)c(-c2cncnc2)c1
InChIKey: YOZVHPXVZXYZOH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Radiometric Filter-Binding Assay: For determination of ABL kinase activity, the radiometric filter-binding assay was used. The assay was performed by mixing 10 μL of the compound pre-diluted with 10 μL of ATP (20 M ATP with 0.1 μCi [γ-33P]-ATP) with the phospho-acceptor peptide poly[Ala6Glu2LysHBr5Tyr1]=polyAEKY) in 20 mM Tris/HCl pH 7.5, 1 mM DTT, 10 mM MgCl2, 0.01 mM Na3VO4, 50 mM NaCl. 10 μL of enzyme (ranging between 5 nM to 20 nM) was added to initiate the reaction. Pre-incubation of enzyme with compounds (when stated) was performed by exposing the enzyme to compounds prior to addition of the substrate mixture (ATP and/or peptide substrate). After 15 min at room temperature, the reaction was stopped by the addition of 50 μL 125 mM EDTA, and the peptide-bound 33P separated on filter-plates (PVDF or MAIP; Millipore, Volketswil, Switzerland) prepared according to the manufacturer's instructions. Filter-plates were washed 3× with 0.5% H3PO4, followed by addition of 30 μL scintillation cocktail (Microscint, Perkin Elmer) per well and then analysed in a TopCount NXT scintillation counter (Perkin Elmer).
Measurement: IC50 = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 463.42, ALogP 2.65, HBA 8, HBD 3, PSA 118.49, QED 0.39
Compound (SMILES): O=C(Cn1cnc2ccc(-c3cccc(OCC(O)CN4CCc5ccccc5C4)c3)cc21)N1CCOCC1
InChIKey: ABTMEYFSZRGPKR-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 = 101 nM (pChEMBL 7)
Descriptors: MW 526.64, ALogP 3.36, HBA 7, HBD 1, PSA 80.06, QED 0.38
Compound (SMILES): COc1cccc(C(=O)N2CCC(Nc3ccc(-c4cc(C)cc(Nc5cc(C(F)(F)F)ccn5)n4)cn3)CC2)c1
InChIKey: ZDSJSLAHJMGQMQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSyk fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-Syk (Carna Biosciences #08-176) (5 pM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 uM final concentration). Final volume of the reaction was 10 uL. Phosphorylation of the peptide was allowed to proceed for 45x at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 562.6, ALogP 6.33, HBA 7, HBD 2, PSA 92.27, QED 0.27
Compound (SMILES): Cc1ccc(N(C(=O)OC[C@H]2CC[C@H](COCC(=O)O)CC2)c2cccc(F)c2)cc1
InChIKey: WWKMUZSOBDQEHP-WGSAOQKQSA-N
Target: Prostacyclin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43119
Assay: Agonist activity at human recombinant prostacyclin receptor expressed in CHO-K1 cells assessed as induction of cAMP accumulation incubated for 1 hr by HTRF assay
Measurement: EC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 429.49, ALogP 5.32, HBA 4, HBD 1, PSA 76.07, QED 0.61
Compound (SMILES): COc1cc(-c2cc(C)ccc2Cl)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: VVVYSTFUJKLTTG-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 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 -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 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 539.97, ALogP 5.56, HBA 7, HBD 1, PSA 103.43, QED 0.3
Compound (SMILES): Cc1cc(C#N)cc(C)c1Oc1cc(Cl)nc(NC2CCN(Cc3ccc(S(C)(=O)=O)cc3)CC2)c1
InChIKey: XFEVJUZGIJRDFS-UHFFFAOYSA-N
Target: Human immunodeficiency virus type 1 reverse transcriptase (SINGLE PROTEIN), Human immunodeficiency virus 1
UniProt: Q72547
Assay: Inhibition of recombinant wild type HIV1 p66/p51 using poly(rA) template/oligo(dT)16 primer after 40 mins by PicoGreen-based spectrofluorometric method
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 525.07, ALogP 5.5, HBA 7, HBD 1, PSA 95.32, QED 0.41
Compound (SMILES): CCOc1ccc2cc(C(=O)NCCc3c[nH]c4ccccc34)c(=O)oc2c1
InChIKey: DZSGSEFRADGHDP-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Electrophorus electricus
UniProt: O42275
Assay: Inhibition of electric eel AChE using acetylthiocholine iodide as substrate measured for 2 mins by Ellman's method
Measurement: IC50 = 43800 nM (pChEMBL 4.360000133514404)
Descriptors: MW 376.41, ALogP 3.65, HBA 4, HBD 2, PSA 84.33, QED 0.5
Compound (SMILES): COc1ccc(C)cc1-c1cc(F)cc(C(CC(=O)O)c2ncc(C)s2)c1
InChIKey: YEGIICXUKYAYRL-UHFFFAOYSA-N
Target: Lysosomal protective protein (SINGLE PROTEIN), Homo sapiens
UniProt: P10619
Assay: Inhibition Assay: Recombinant human cathepsin A (residues 29-480, with a C-terminal 10-His tag; R&D Systems, #1049-SE) was proteolytically activated with recombinant human cathepsin L (R&D Systems, #952-CY). Briefly, cathepsin A was incubated at 10 ug/ml with cathepsin L at 1 ug/ml in activation buffer (25 mM 2-(morpholin-4-yl)-ethanesulfonic acid (MES), pH 6.0, containing 5 mM dithiothreitol (DTT)) for 15 min at 37° C. Cathepsin L activity was then stopped by the addition of the cysteine protease inhibitor E-64 (N-(trans-epoxysuccinyl)-L-leucine-4-guanidinobutylamide; Sigma-Aldrich, # E3132; dissolved in activation buffer/DMSO) to a final concentration of 10 uM.The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature.
Measurement: IC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 385.46, ALogP 5.18, HBA 4, HBD 1, PSA 59.42, QED 0.63
Compound (SMILES): O=C1c2cc3c(cc2C(=O)N1CCC1CCN(Cc2ccccc2)CC1)OCO3
InChIKey: JXGLOHWZAFNLOQ-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Mus musculus
UniProt: P21836
Assay: In Vitro Pharmacodynamic Assay: Modified Ellman method (Alvin V. et al. JWS-USC-75-IX Improves Information Processing and Cognitive Function in Animal Models[J]. Journal of Pharmacology and experimental therapeutics, 2010, 336(3): 751) was employed in the test, for testing the inhibition effect of a series of compounds of formula (I) on acetylcholinesterase, so that active AchEIs were screened out, and their activity were evaluated via the IC50 value of inhibition of the series of compounds of formula (I) on the enzyme.
Measurement: IC50 = 86 nM (pChEMBL 7.070000171661377)
Descriptors: MW 392.46, ALogP 3.31, HBA 5, HBD 0, PSA 59.08, QED 0.73
Compound (SMILES): O=c1cc(CCc2cccc(Cl)c2)c2cc(C(c3ccc(Cl)cc3)c3nccs3)ccc2[nH]1
InChIKey: QRHVHCOQSXTVEG-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: The following mixtures and buffer solutions were prepared: (a) Buffer 1: HBSS (Mediatech Cat#21-023-CV) with 5 mM HEPES (1 mM stock, Gibco BRL Cat#15630-056) and 0.1% BSA (7.5% stock, Invitrogen Cat#15260-037); (b) Buffer 2: 0.5 mM IBMX (200 mM stock in DMSO, Sigma 15879) in Buffer 1; (c) 1 uM cAMP Standard (50 uM stock, Perkin Elmer Cat# AD0262) diluted in Buffer 2 and serially diluted in Buffer 2, 12 doses at 1/2 dilutions resulting in a dose range of 1 uM to 0.5 nM; (d) d2 labelled cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 6 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); (e) anti-cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 5 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); and (f) Forskolin (Sigma Cat# F6886, 10 mM in DMSO) diluted first in DMSO to 1 mM and then to 1.5 uM in Buffer 2.
Measurement: EC50 = 121 nM (pChEMBL 6.920000076293945)
Descriptors: MW 491.44, ALogP 7.26, HBA 3, HBD 1, PSA 45.75, QED 0.27
Compound (SMILES): CC(Nc1nc(NC2CC2)n2ncc(/C=C3\NC(=O)NC3=O)c2n1)c1cccs1
InChIKey: OOVSMCOQSSYNQR-GHXNOFRVSA-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 410.46, ALogP 2.11, HBA 9, HBD 4, PSA 125.34, QED 0.36
Compound (SMILES): CN1CC(NC(=O)c2ccc3cc(Oc4ccc(C(F)F)cn4)ccc3n2)CC1=O
InChIKey: SWTSCRJWTGVILO-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 947 nM (pChEMBL 6.019999980926514)
Descriptors: MW 412.4, ALogP 3.32, HBA 5, HBD 1, PSA 84.42, QED 0.7
Compound (SMILES): Fc1ccc(C2OCCc3c2[nH]c2ccccc32)cc1Cl
InChIKey: IVHNQGZPWYMIRE-UHFFFAOYSA-N
Target: Sodium/iodide cotransporter (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q63008
Assay: Inhibition of sodium iodide symporter in rat FRTL5 cells assessed as decrease in [125I]-sodium iodide uptake after 60 mins by scintillation counting method
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 301.75, ALogP 4.62, HBA 1, HBD 1, PSA 25.02, QED 0.7
Compound (SMILES): CNC(=O)c1cc(Nc2ncnc(N3CC(Oc4ccc(C(F)(F)F)cc4)C3)n2)ccc1F
InChIKey: XTAYGNQXKUWVPX-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 = 210 nM (pChEMBL 6.679999828338623)
Descriptors: MW 462.41, ALogP 3.4, HBA 7, HBD 2, PSA 92.27, QED 0.54
Compound (SMILES): COc1ccc(-c2cccc(F)c2F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: HWGYZFJFZCOVSA-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 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 -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 = 118 nM (pChEMBL 6.929999828338623)
Descriptors: MW 509.49, ALogP 4.73, HBA 7, HBD 1, PSA 103.43, QED 0.35
Compound (SMILES): NS(=O)(=O)c1ccccc1NS(=O)(=O)c1cc(Cl)c(Cl)s1
InChIKey: GHHIWTKGUAFFNW-UHFFFAOYSA-N
Target: Prostaglandin E synthase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7Z7
Assay: Biochemical Assay: A solution of test compound was added to a diluted microsome preparation containing human mPGES-1 and pre-incubated for 15 minutes in potassium phosphate buffer pH 6.8 with cofactor glutathione (GSH). Corresponding solutions without test compound were used as positive controls, and corresponding solutions without test compound and without microsomes were used as negative controls. The enzymatic reaction was then started by addition of the substrate PGH2 in an organic solution (dry acetonitrile).The typical reaction conditions of the enzymatic reaction were thus: Test compound: ranging from 60 uM to 0.002 uM, or zero in positive and negative controls; potassium phosphate buffer pH 6.8: 50 mM; GSH: 2.5 mM; mPGES-1-containing microsomes: 2 ug/mL (sample and positive controls) or 0 ug/mL (negative control); PGH2: 10.8 uM; Acetonitrile: 7.7% (v/v); DMSO: 0.6% (v/v). The reaction was stopped after one minute by adding an acidic solution (pH 1.9) of ferric chloride.
Measurement: IC50 = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 387.29, ALogP 2.5, HBA 5, HBD 2, PSA 106.33, QED 0.84
Compound (SMILES): c1nc(N2CCOCC2)c2c(-c3cn[nH]c3)c[nH]c2n1
InChIKey: XJYBPVJUBUUJKF-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Lantha Screen Technology Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat # PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: 3 uL of a working solution of substrate (233 nM LRRKtide, 117 uM ATP) prepared in assay buffer (50 mM HEEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to a low volume Greiner 384-well plate. The compound dose response was prepared by diluting compound to a top concentration of 3.16 mM in 100% DMSO and serial diluted by half-log in DMSO 11 times. Aliquots (3.5 uL) of the 100% DMSO dose response were mixed with 46.5 uL water.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 270.3, ALogP 1.18, HBA 5, HBD 2, PSA 82.72, QED 0.73
Compound (SMILES): COc1cc(OC)cc(N(CCN2CCCC(N)C2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: WTEYUYQDYILCNS-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 354.81 nM (pChEMBL 6.449999809265137)
Descriptors: MW 487.61, ALogP 3.61, HBA 9, HBD 1, PSA 94.56, QED 0.4
Compound (SMILES): CC1Cc2c(nnn2-c2ccccn2)CN1C(=O)c1cccc(Cl)c1Cl
InChIKey: LNFMAINDAKFZDA-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Radioligand Binding Assay: human or rat P2X7-1321 N1 cells were collected and frozen @−80° C. On the day of the experiment, cell membrane preparations were made according to standard published methods. The total assay volume was 100 μl: 10 μl compound (10×)+(b) 40 μl tracer (2.5×)+50 μl membrane (2×). The tracer used for the assay was tritiated A-804598. The compound can be prepared as described in the literature. (Donnelly-Roberts, D. Neuropharmacology 2009, 56 (1), 223-229.) Compounds, tracer and membranes were incubated for 1 hour @ 4° C. The assay was terminated by filtration (GF/B filters pre-soaked with 0.3% PEI) and washed with washing buffer (Tris-HCl 50 mM).
Measurement: Ki = 6.3 nM (pChEMBL 8.199999809265137)
Descriptors: MW 388.26, ALogP 3.56, HBA 5, HBD 0, PSA 63.91, QED 0.67
Compound (SMILES): CN1C(=O)C2(N=C1N)c1cc(-c3cccc(C#N)c3)ccc1C[C@]21CC[C@@H](N(C)C)CC1
InChIKey: ZKMXGRISRJAUBG-AHQGXOPPSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.435): For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 ul OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 ul water containing the desired concentration of test compound and 1% DMSO, substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were given to the plate followed by the addition of 10 ul of BACE.
Measurement: IC50 = 13500 nM (pChEMBL 4.869999885559082)
Descriptors: MW 427.55, ALogP 3.25, HBA 5, HBD 1, PSA 85.72, QED 0.8
Compound (SMILES): Cn1cc(-c2ccc3[nH]nc(-c4cc(N5CCC(F)(F)C5)ncn4)c3c2)cn1
InChIKey: SGEHWCFRIGXRTH-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 3.7 nM (pChEMBL 8.430000305175781)
Descriptors: MW 381.39, ALogP 3.27, HBA 6, HBD 1, PSA 75.52, QED 0.59
Compound (SMILES): CC(C)CN([C@@H](C)c1ccc(N2CCN(S(=O)(=O)N(C)C)CC2)cc1)S(=O)(=O)Cc1ccccc1
InChIKey: WXZUGHCHUOFZKW-QFIPXVFZSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Assay: On day of the assay, 100 uL of 0.05% CHAPS (in deionized H2O) was added to all wells of the GFB Unifilter plate and allowed soak for 1 h. A wash buffer of 50 mM HEPES (pH 7.4), 150 mM NaCl, and 5 mM MgCl2 was prepared to wash the filter plate. To prepare an assay buffer, BSA was added to the wash buffer to reach 0.01% and DTT was added to reach 1 mM. For IC50 mode, 10 mM compound stocks were serially diluted in DMSO with DMSO to give 20x required final concentration in DMSO (15 uL compound+30 uL DMSO). The 20x compound stocks were diluted in DMSO with Assay Buffer 4-fold to reach 5x the final test concentration in 25% DMSO (10 uL compound+30 uL Assay Buffer). Solutions were mixed by aspiration several times with a pipette set on 50 uL volume. For the assay, 10 uL of 5x compound stock solutions in 25% DMSO were added to the assay plate in duplicate.
Measurement: IC50 = 3700 nM (pChEMBL 5.429999828338623)
Descriptors: MW 522.74, ALogP 3.16, HBA 5, HBD 0, PSA 81.24, QED 0.48
Compound (SMILES): Cc1nc2cccc(-c3cc4c([nH]3)CCNC4=O)c2nc1N[C@H]1CCCN(C(=O)OC(C)(C)C)C1
InChIKey: RXAMEJTTXXXMKB-INIZCTEOSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 384-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 191 nM (pChEMBL 6.71999979019165)
Descriptors: MW 476.58, ALogP 4.03, HBA 6, HBD 3, PSA 112.24, QED 0.53
Compound (SMILES): Cc1nc(-c2ccncc2)sc1C(=O)Nc1cn2nc(-c3c(-c4ccc(F)cc4)nn(C)c3C3CCNCC3)ccc2n1
InChIKey: VSQBEMDBWISNFJ-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 ul prepared from 15 ul additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 ul of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIPÂ®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 uM ATP, 1.5 uM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 593.69, ALogP 5.48, HBA 10, HBD 2, PSA 114.92, QED 0.26
Compound (SMILES): Cc1cccc(C)c1N1C[C@]2(C(=O)Nc3cc(C(F)(F)F)cc(C(F)(F)F)c3)CCO[C@@H]2C1=O
InChIKey: KAZXVVJFETXMIN-WIYYLYMNSA-N
Target: Chemerin-like receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99788
Assay: Displacement of [125I]-chemerin from ChemR23 (unknown origin) transfected in mouse BaF3 cells after 3 hrs by scintillation counting method
Measurement: IC50 <= 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 486.41, ALogP 5.1, HBA 3, HBD 1, PSA 58.64, QED 0.61
Compound (SMILES): CCN(CC)CCOc1ccc(/C(=C(/CCCO)c2ccccc2)c2ccc(O)cc2)cc1.Cl
InChIKey: AGDKCEPOEPTHOT-FJBFXRHMSA-N
Target: Estrogen-related receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P62508
Assay: Inhibition of fluorescien-conjugated coactivator PGC1a binding to GST-tagged human ERRgamma LBD after 1 hr by TR-FRET assay
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 445.6, ALogP 5.84, HBA 4, HBD 2, PSA 52.93, QED 0.34
Compound (SMILES): O=C(Nc1cn(CC(=O)N2C[C@H]3CN(CC4CCC(F)(F)CC4)C[C@H]3C2)nc1-c1cc(Cl)ccc1OC(F)F)c1cnn2cccnc12
InChIKey: DJOBATCLQYMVDJ-OYRHEFFESA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.66 nM (pChEMBL 9.180000305175781)
Descriptors: MW 689.11, ALogP 5.32, HBA 9, HBD 1, PSA 109.89, QED 0.24
Compound (SMILES): CCOC(=O)N1CCC(S(=O)(=O)c2c(Cl)ccc(NC(=O)Nc3ccccc3Oc3ccccc3)c2O)CC1
InChIKey: NEJBAMDKVNDHJM-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P25024
Assay: Displacement of [125I]IL8 from recombinant human CXCR1 expressed in CHO cell membranes measured after 1 hr by TopCount scintillation counting method
Measurement: IC50 < 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 574.06, ALogP 5.88, HBA 7, HBD 3, PSA 134.27, QED 0.3
Compound (SMILES): C[C@H](Nc1ncnc(N)c1Cl)c1nc2cccc(Cl)c2c(=O)n1-c1cnc(N)cn1
InChIKey: ZUYRSTSZRRJDEM-QMMMGPOBSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The activities were measured using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. TR-FRET monitored the formation of 3,4,5-inositol triphosphate molecule that competed with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidylinositide 3-phosphate product resulted in a decrease in TR-FRET signal as the labeled fluorophore was displaced from the GRP-1 protein binding site. Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (75-500 uM), 2 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 uM, and PI3Kγ at 2 nM. The compounds of Table 1 and Compound X ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) and Compound Y ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window). The results were normalized based on positive (1 uM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3K α, β, δ and γ were calculated from the fit of the dose-response curves to a four-parameter equation.
Measurement: IC50 = 570 nM (pChEMBL 6.239999771118164)
Descriptors: MW 444.29, ALogP 2.61, HBA 10, HBD 3, PSA 150.52, QED 0.43
Compound (SMILES): Nc1ncnc2c1c(-c1ccc(NC(=O)C3(C(=O)Nc4ccc(F)cc4)CC3)cc1)cn2C1CC(CN2CCCC2)C1
InChIKey: USFSEQYCEUYNDG-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 567.67, ALogP 5.22, HBA 7, HBD 3, PSA 118.17, QED 0.25
Compound (SMILES): CNc1nnc(-c2ccc3[nH]cc(-c4cccc(-n5cnc(C)c5)n4)c3c2)s1
InChIKey: OLKXVDKTEYJKPA-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 13.24 nM (pChEMBL 7.880000114440918)
Descriptors: MW 387.47, ALogP 4.28, HBA 7, HBD 2, PSA 84.31, QED 0.48
Compound (SMILES): COc1cc(N2CCOCC2)ccc1Nc1nc2c(c(Nc3ccccc3S(=O)(=O)NCCN(C)C)n1)C(C)(C)CN2
InChIKey: BKZOKZDNJWKUMZ-UHFFFAOYSA-N
Target: Focal adhesion kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q05397
Assay: Biochemical Assay: To measure inhibitory activity of FAK/PYK2 inhibitors, compounds were first prepare as a 1 mM stock in 100% DMSO and 3-fold serial dilution was performed in a 96-well plate (Corning, 3897) to generate 12 different concentration of 100× stock. A 5 μl of 100× stock of each concentration was added to wells containing 95 μl of 1× reaction buffer (40 mM Tris, pH7.5, mM MgCl2, 1 mM DTT and 1 mM CHAPS) to generate 5× stock. Then 2 μl of 5× stock of each concentration was added to a 384 wells-OptiPlate (PerkinElmer, 6007299). For FAK, 4 μl of 2.5 nM FAK, 1 μl of 8 μg/μl BSA and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 33.3 μM ATP. The reaction was allowed to proceed for 120 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). For PYK2, 5 μl of 2 nM PYK2 and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 46.6 μM ATP. The reaction was allowed to proceed for 40 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). Upon quenching of the reaction, 5 μl of 8 nM anti-phosphotyrosine antibody was added to each well and incubated for 60 minutes.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 596.76, ALogP 3.35, HBA 11, HBD 4, PSA 132.98, QED 0.26
Compound (SMILES): NC(=O)c1cc(N2C[C@H](O)[C@@H](O)C2)nc(-c2ccc(Oc3ccc(F)cc3)cc2)n1
InChIKey: DQVWZYVJOHZANB-ROUUACIJSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FLIPR Assay: Assay Buffer: The assay buffer was formulated by removing 120 mL from a 1 L bottle of fresh, sterile dH2O (Mediatech, Herndon, Va.) and adding 100 mL of 10×HBSS that does not contain Ca++ or Mg++ (Gibco, Invitrogen, Grand Island, N.Y.) followed by 20 mL of 1.0 M Hepes, pH 7.3 (Fisher Scientific, BP299-100). The final buffer consisted of 20 mM Hepes, pH 7.3, 1.261 mM CaCl2, 0.493 mM MgCl2, 0.407 mM Mg(SO)4, 5.33 mM KCl, 0.441 mM KH2PO4, 137 mM NaCl, 0.336 mM Na2HPO4 and 0.556 mM D-glucose (Hanks et al., Proc. Soc. Exp. Biol. Med. 71:196 (1949)), and the simple formulation was typically the basic buffer throughout the assay (i.e., all wash and addition steps).
Measurement: IC50 = 410 nM (pChEMBL 6.389999866485596)
Descriptors: MW 410.41, ALogP 1.72, HBA 7, HBD 3, PSA 121.8, QED 0.59
Compound (SMILES): COc1cc(Cl)cnc1C(=O)Nc1cc(F)c(F)c([C@]2(C)C[C@@H](C(F)(F)F)OC(N)=N2)c1
InChIKey: PSHOVOIOCRZCGO-UGSOOPFHSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE FRET Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 71 nM (pChEMBL 7.150000095367432)
Descriptors: MW 478.81, ALogP 4.16, HBA 6, HBD 2, PSA 98.83, QED 0.64
Compound (SMILES): C=CC(=O)Nc1ccccc1Nc1cc(N(C)C(=O)Nc2cccc(OC)c2Cl)ncn1
InChIKey: RARQZNWPZNZGAG-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Kinase HotSpotâ¿¢ Assay: Compounds were profiled for FGFR inhibition activity at Reaction Biology Corporation (Malvern, Pa.) with their Kinase HotSpot assay. See, Anastassiadis et al., 2011, Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29, 1039-1045.Recombinant FGFR1 (2.5 nM), FGFR2 (1 nM), FGFR3 (5 nM), or FGFR4 (12 nM) (Invitrogen) was prepared as a mixture with substrate KKKSPGEYVNIEFG (SEQ ID NO:1) (20 uM, FGFR1 substrate); and Poly [E,Y]4:1 (0.2 mg/ml, FGFR2,3,4 substrate)] in kinase reaction buffer (20 mM HEPES-HCl, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 1 mM EGTA, 0.02% Brij35, 0.1 mM Na3VO4, 0.02 mg/ml BSA, 2 mM DTT, and 1% DMSO). Compound was added to the enzyme/substrate mixture using acoustic technology (Labcyte Echo 550, Sunnyvale, Calif.) (see, Olechno et al., 2006, Improving IC50 results with acoustic droplet ejection. JALA 11, 240-246) and pre-incubated for 0, 15, or 60 minutes at room temperature. After compound pre-incubation, a mixture of ATP (Sigma-Aldrich) and 33P-γ-ATP (PerkinElmer) was added to a final concentration of 10 uM to initiate kinase reactions. Reactions were incubated for 120 minutes at room temperature and then spotted onto Whatman P81 ion exchange filter paper. Unbound phosphate was removed by extensively washing filters in 0.75% phosphoric acid. See, Anastassiadis et al., 2011, Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29, 1039-1045.
Measurement: IC50 = 673 nM (pChEMBL 6.170000076293945)
Descriptors: MW 452.9, ALogP 4.68, HBA 6, HBD 3, PSA 108.48, QED 0.45
Compound (SMILES): Cc1c([C@@H](O)CN2CCCC3(CCCN(C[C@H](O)c4ccc5c(c4C)COC5=O)C3)C2)ccc2c1COC2=O
InChIKey: YJWJJJTWMMJKEG-PWDXKWARSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Thallium Flux Assay: The ROMK channel functional thallium flux assay is performed in 384 wells, using the FLIPR-Tetra instrument. HEK-hKir1.1 cells are seeded in Poly-D-Lysine microplates and kept in a 37° C.-10% CO2 incubator overnight. On the day of the experiment, the growth media is replaced with the FluxOR reagent loading buffer and incubated, protected from light, at ambient temperature (23-25° C.) for 90 min. The loading buffer is replaced with assay buffer test compound followed by 30 min incubation at ambient temperature, where the Thallium/Potassium stimulant is added to the microplate. Step Protocol 1. Seed HEK-hKir1.1 cells (50 ul at 20,000 cells/well) in 384-well PDL coated Microplates 2. Allow cells to adhere overnight in humidified 37° C./10% CO2 incubator 3. Completely remove cell growth media from microplate and replace with 25 ul loading buffer 4. Incubate Microplate at room temperature, protected form light, for 90 min 5. Remove loading buffer and replace with 25 ul 1x Assay BufferÂ±test compound.Â  6. Incubate microplate at room temperature, protected form light, for 30 min 7. At FLIPR-Tetra 384: Add stimulant (Thallium/Potassium) solution to microplate and monitor fluorescence. Excitation=400 nm, Emission=460 & 580 nm. Collect data for 10 min.Â  Data Calculation: The fluorescence intensity of wells containing 3 uM of a standard control ROMK inhibitor of the present invention is used to define the ROMK-sensitive component of thallium flux. Fluorescence in the presence of test compounds is normalized to control values to provide % fluorescence change. IC50 values represent the concentration of compound that inhibits 50% of the ROMK thallium flux signal.
Measurement: IC50 = 358.6 nM (pChEMBL 6.449999809265137)
Descriptors: MW 534.65, ALogP 3.59, HBA 8, HBD 2, PSA 99.54, QED 0.54
Compound (SMILES): C[C@@]1(c2sc(Br)cc2Cl)CS(=O)(=O)C2(CCC2)C(=N)N1
InChIKey: BFFXPMHURKDIDG-NSHDSACASA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme Inhibition of BACE1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.Varying concentrations of inhibitors at 3x the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 ul) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol.
Measurement: Ki = 1566 nM (pChEMBL 5.800000190734863)
Descriptors: MW 397.75, ALogP 3.3, HBA 4, HBD 2, PSA 70.02, QED 0.76
Compound (SMILES): CC#Cc1cccc(-c2ccc3c(c2)C2(N=C(C)C(N)=N2)[C@]2(CC[C@H](OC)CC2)C3)c1Cl
InChIKey: OWDYFPYKCCMOHX-MJPADRBUSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: TR-FRET Assay: The β-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human β-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc 10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in −80° C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 μg/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 μM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 μl) was added to the plate then 1 μl of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes. Substrate (10 μl) was then added, mixed and the reaction proceeded for 15 minutes at r.t. The reaction was stopped with the addition of Stop solution (7 μl, NaAcetate, pH 9). The fluorescence of the product was measured on a Victor II plate reader with an excitation wavelength of 340 nm and an emission wavelength of 615 nm. The assay was performed in a Costar 384 well round bottom, low volume, non-binding surface plate (Corning #3676). The final concentration of the enzyme was 2.7 g/ml; the final concentration of substrate was 100 nM (Km of 250 nM). The dimethylsulphoxide control, instead of test compound, defined the 100% activity level and 0% activity was defined by wells lacking enzyme (replaced with reaction buffer). A control inhibitor was also used in dose response assays and had an IC50 of 150 nM.
Measurement: IC50 = 189 nM (pChEMBL 6.71999979019165)
Descriptors: MW 445.99, ALogP 5.49, HBA 4, HBD 1, PSA 59.97, QED 0.62
Compound (SMILES): CC(C)(C)c1cnc(CSc2cnc(NC(=O)CCCCC(=O)NO)s2)o1
InChIKey: SNMMJBCMTCRCSM-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: Inhibition of C-terminal His6-tagged full length CDK2/N-terminal GST-tagged recombinant full length human cyclin E1 expressed in baculovirus infected SF21 insect cells using histone H1 as substrate after 2 hrs in presence of [gamma-33P]-ATP
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 412.54, ALogP 3.73, HBA 8, HBD 3, PSA 117.35, QED 0.25