lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CS(=O)(=O)CC(=O)Nc1c2c(nn1CCCOC(F)F)C[C@]1(CCc3cc(OCC(F)(F)F)ccc31)NC2=O
InChIKey: XVZIXBQZWOPEEN-QFIPXVFZSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: Inhibitory Activity: Solutions of the compounds of the present invention in DMSO were each aliquoted into 0.2-μL portions in a 384-well polystyrene microplate produced by Corning Incorporated, and 5 μL of an enzyme solution prepared with an assay buffer (100 mmol/L phosphate buffer (pH 7.4) containing 2 mmol/L DTT) and 5 μL of a substrate solution (100 mmol/L phosphate buffer (pH 7.4), 30 μmol/L 2-Oleoylglycerol, 10 μmol/L Oleoyl-CoA) were added thereto, and the resultant was stirred and centrifuged, and incubated in a moist chamber at room temperature for 1 hour. After enzymatic reaction, 50 μL of a quenching solution (containing 0.2 μmol/L Diolein-d5, 0.4% formic acid, and 50% isopropanol) containing Internal Standard (IS) was added to terminate the reaction, and the resultant was sealed in a plate produced by Shimadzu GLC Ltd., and then stirred and centrifuged, and measurement was performed by using an electrospray ionization method with a RapidFire360 and Agilent 6550 Q-TOF mass spectrometer. Diolein as a reaction product (P) of 2-Oleoylglycerol as the substrate and an ammonium adduct ion of the IS were detected, and the peak intensity ratio, P/IS, was calculated from the peak heights to evaluate the inhibitory activity. Inhibitory activities with/without addition of enzyme were defined as Control (+)/Control (−), respectively, and the respective % inhibitions were defined as 0% inhibition and 100% inhibition. The inhibitory activity was calculated from formula below with TIBCO Spotfire (produced by TIBCO Software Inc.):Inhibitory activity (%)=[1−{Sample−Control(−)}/{Control(+)−Control(−)}]*100where Sample indicates a peak intensity ratio: P/IS, when the compound of the present invention was added.
Measurement: IC50 = 1.34 nM (pChEMBL 8.869999885559082)
Descriptors: MW 580.53, ALogP 2.56, HBA 8, HBD 2, PSA 128.62, QED 0.33
Compound (SMILES): CCc1nc2c(-c3c[nH]cn3)cc(N3CCOCC3)cc2n1-c1ccnc2c(Cl)cccc12
InChIKey: HWMNYRIZEVLQBC-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: TR-FRET PI3K Beta Assay: 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 2× Km ATP (75-500 μM), 2 μM 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 μM, and PI3Kγ at 2 nM. The compounds 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 μs delay and 500 μs read window).The results were normalized based on positive (1 μM 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. These assays generally produced results within 3-fold of the reported mean. As shown in Table 1, the compounds provided herein are inhibitors of PI3Kβ.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 458.95, ALogP 5.02, HBA 6, HBD 1, PSA 71.86, QED 0.41
Compound (SMILES): [C-]#[N+]c1ccc(-c2nc(N3CCC(N)CC3)n(C)c(=O)c2-c2ccc(COC)cc2)cc1F
InChIKey: FHNHWPOYNURJKW-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: In Vitro Enzyme Inhibition Assay: This assay determines the ability of a test compound to inhibit LSD1 demethylase activity. E. coli expressed full-length human LSD1 (Accession number O60341) was purchased from Active Motif (Cat#31334).The enzymatic assay of LSD1 activity is based on Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The inhibitory properties of compounds to LSD1 were determined in 384-well plate format under the following reaction conditions: 0.1-0.5 nM LSD1, 50 nM H3K4me1-biotin labeled peptide (Anaspec cat #64355), 2 μM FAD in assay buffer of 50 mM HEPES, pH7.3, 10 mM NaCl, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-unmodified histone H3 lysine 4 (H3K4) antibody (PerkinElmer) in the presence of LSD1 inhibitor such as 1.8 mM of Tranylcypromine hydrochloride (2-PCPA) in LANCE detection buffer (PerkinElmer) to final concentration of 12.5 nM and 0.25 nM respectively.The assay reaction was performed according to the following procedure: 2 μL of the mixture of 150 nM H3K4me1-biotin labeled peptide with 2 μL of 11-point serial diluted test compound in 3% DMSO were added to each well of plate, followed by the addition of 2 μL of 0.3 nM LSD1 and 6 μM of FAD to initiate the reaction. The reaction mixture was then incubated at room temperature for one hour, and terminated by the addition of 6 μL of 1.8 mM 2-PCPA in LANCE detection buffer containing 25 nM Phycolink Streptavidin-allophycocyanin and 0.5 nM Europium-anti-unmodified H3K4 antibody. Enzymatic reaction is terminated within 15 minutes if 0.5 LSD1 enzyme is used in the plate. Plates were read by EnVision Multilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 447.51, ALogP 3.88, HBA 6, HBD 1, PSA 77.74, QED 0.6
Compound (SMILES): O=C(NO)c1cc(NC(=O)c2c(O)[nH]c(=O)[nH]c2=O)ccn1
InChIKey: FOYYEXMCHXAYII-UHFFFAOYSA-N
Target: Xanthine dehydrogenase/oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P47989
Assay: Bioactivity Assay: Xanthine oxidase inhibition was determined using a standard fluorescence-based assay for xanthine oxidase activity (McHale A, Grimes H, Coughlan M P: Int J Biochem. 10:317-9, 1979) with minor variations. The procedure was internally standardized using allopurinol and DPI as controls for all experiments after determination of their optimal inhibitory concentrations. Experiments on test compounds were performed in triplicate in multi-well plates using 10 concentrations of each compound that ranged over a 3-fold dilution.
Measurement: IC50 = 73800 nM (pChEMBL 4.130000114440918)
Descriptors: MW 307.22, ALogP -1.46, HBA 7, HBD 6, PSA 177.27, QED 0.3
Compound (SMILES): CC(=O)N1CCN(Cc2nnc(-c3ccc4[nH]c(-c5ccnc6[nH]ncc56)c(C(C)C)c4c3)o2)CC1
InChIKey: GWVLCUPAVAQENE-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κc Band AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Bue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 1570 nM (pChEMBL 5.800000190734863)
Descriptors: MW 484.56, ALogP 3.94, HBA 7, HBD 2, PSA 119.83, QED 0.39
Compound (SMILES): Oc1ccc(C(O)CCc2ccccc2O)c(O)c1
InChIKey: RASKQZARLSAQOE-UHFFFAOYSA-N
Target: Polyphenol oxidase 1 (SINGLE PROTEIN), Agaricus bisporus
UniProt: Q00024
Assay: Tyrosinase Inhibition Assay: A tyrosinase inhibition assay was carried out using the method reported by Jones et al. (2002) Pigment. Cell Res. 15:335. Using this method, the conversion of L-Dopa, a substrate of tyrosinase, into dopachrome is followed by monitoring absorption at 450 nm. Tyrosinase was prepared in 50 mM potassium phosphate buffer, pH 6.8 (assay buffer) at 2000 U/ml and stored at −20° C. in 1 ml aliquots prior to use. For use in assays, stock enzyme solutions were thawed and diluted to 200 U/ml with assay buffer. A 2 mM working solution of substrate, L-DOPA, was prepared in assay buffer for each assay. Samples were dissolved in 10% DMSO (0.5 ml) and diluted to 5 ml with assay buffer. The reaction mixture consisted of 0.050 ml 2 mM L-DOPA, 0.050 ml 200 U/ml mushroom tyrosinase and 0.050 ml inhibitor. Reaction volume was adjusted to 200 μl with assay buffer. Assays were performed in 96 well Falcon 3097 flat-bottom microtiter plates (Beckton Dickinson, N.J.). Appearance of dopachrome was measured with a WALLAC 1420 Multilable Counter (Turku, Finland). Average velocity was determined from linear enzyme rate as measured by change in absorbance (ΔA450) at 450 nm per minute.
Measurement: IC50 = 700 nM (pChEMBL 6.159999847412109)
Descriptors: MW 260.29, ALogP 2.47, HBA 4, HBD 4, PSA 80.92, QED 0.68
Compound (SMILES): O=C(c1cccc(-c2nc(NC(P(=O)(O)O)P(=O)(O)O)c3ccsc3n2)c1)N1CCC(c2ccccc2)CC1
InChIKey: YGYNQQXBLWLNEX-UHFFFAOYSA-N
Target: Geranylgeranyl pyrophosphate synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O95749
Assay: In Vitro hGGPPS Inhibition Assay: The assay was based on a literature procedure (Kavanagh, et al. J. Biol. Chem., 2006, 281, 22004-22012) with minor modifications. All assays were run in triplicate using recombinant human GGPPS (80 ng), FPP (10 μM), IPP (8.3 μM; 3H-IPP, 40 mCi/mmoL) in a final volume of 100 μL buffer containing 50 mM Tris pH 7.7, 2 mM MgCl2, 1 mM TCEP, 5 μg/mL BSA and 0.2% (w/v) Tween 20. The enzyme and test compound were pre-incubated in the assay buffer in a volume of 80 μL at 37° C. for 10 mins. Afterwards, the substrates (FPP, IPP) were added to start the reaction, which also bring the compound, substrate, and buffer contents to the desired final concentrations as indicated above. The assay mixture was then incubated at 37° C. for 15 mins (Note: the incubation time is based from the curve determined each time a new batch of enzyme is produced). Assays were terminated by the addition of 200 μL of HCl/MeOH (1:4) and incubated for 10 min at 37° C. The mixture was then extracted with 700 μL of petroleum ether, dried through a plug of anhydrous Mg2SO4 and 300 μL of the dried ligroin phase was combined with 8 mL of scintillation cocktail. Finally, the radioactivity was counted using a Beckman Coulter LS6500 liquid scintillation counter.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 588.52, ALogP 4.43, HBA 7, HBD 5, PSA 173.18, QED 0.19
Compound (SMILES): O=c1[nH]c(CS[C@H]2CC[C@H](O)CC2)nc2cc(OC[C@H]3CCOC3)cc(F)c12
InChIKey: AEUJLXWQVADMCV-YDHLFZDLSA-N
Target: Protein mono-ADP-ribosyltransferase PARP14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q460N5
Assay: Enzymatic Inhibition Assay: Enzymatic inhibition of PARP14 was measured using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) monitoring the auto-modification of PARP14 by biotinylated nicotinamide adenine dinucleotide (biotin-NAD). 1 μL of a dose response curve of each test compound was spotted in 384-well nickel-coated white microplates (Thermo) using a Mosquito (TTP Labtech). Reactions were performed in a 50 μL volume by adding 40 μL of PARP14 in assay buffer (20 mM HEPES pH==8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002%0 Tween20), incubating with test compound at 25° C. for 30 min, then adding 10 μL of biotin-NAD (Biolog). The final concentrations of PARP14 and biotin-NAD are 50 nM and 3 μM, respectively. Reactions proceeded at 25° C. for 3 h, then were quenched with 5 μL of 10 mM unmodified nicotinamide adenine dinucleotide (Sigma-Aldrich). The quenched reactions were washed 3 times with 100 μL of TBST wash buffer (50 mM Tris-HCl, 150 mM NaCl and 0.1% Tween20). Next, to the washed and dried plate was added 25 μL of DELFIA Europium-N1 streptavidin (Perkin Elmer) diluted in DELFIA assay buffer (Perkin Elmer). After a 30 min incubation at 25° C., the plate was washed 5 times with TBST wash buffer. Finally, 25 μL of DELFIA enhancement solution was added. After a 5 min incubation the plate was read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 340 nm and emission of 615 nm to measure the amount of Europium present in each well, informing on the amount of biotin-NAD that was transferred in the automodification reaction.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 408.5, ALogP 3.01, HBA 6, HBD 2, PSA 84.44, QED 0.76
Compound (SMILES): O=C(Nc1cccc(C2CCCN(c3ncnc4[nH]ccc34)C2)c1)Nc1cccnc1O
InChIKey: CJVRTWQCPGAYMO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In Vitro BTK Kinase Assay: The purpose of the BTK in vitro assay is to determine compound potency against BTK through the measurement of IC50. Compound inhibition is measured after monitoring the amount of phosphorylation of a fluorescein-labeled polyGAT peptide (Invitrogen PV3611) in the presence of active BTK enzyme (Upstate 14-552), ATP, and inhibitor. The BTK kinase reaction was done in a black 96 well plate (costar 3694). For a typical assay, a 24 μL aliquot of a ATP/peptide master mix (final concentration; ATP 10 μM, polyGAT 100 nM) in kinase buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 200 μM Na3PO4, 5 mM DTT, 0.01% Triton X-100, and 0.2 mg/ml casein) is added to each well. Next, 1 μL of a 4-fold, 40× compound titration in 100% DMSO solvent is added, followed by adding 15 uL of BTK enzyme mix in 1× kinase buffer (with a final concentration of 0.25 nM). The assay is incubated for 30 minutes before being stopped with 28 μL of a 50 mM EDTA solution. Aliquots (5 μL) of the kinase reaction are transferred to a low volume white 384 well plate (Corning 3674), and 5 μL of a 2× detection buffer (Invitrogen PV3574, with 4 nM Tb-PY20 antibody, Invitrogen PV3552) is added. The plate is covered and incubated for 45 minutes at room temperature. Time resolved fluorescence (TRF) on Molecular Devices M5 (332 nm excitation; 488 nm emission; 518 nm fluorescein emission) is measured. IC50 values are calculated using a four parameter fit with 100% enzyme activity determined from the DMSO control and 0% activity from the EDTA control.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 429.48, ALogP 4.09, HBA 6, HBD 4, PSA 119.06, QED 0.39
Compound (SMILES): CN1CCN(C(=O)c2ccc(CN(C(=O)c3cc(-c4cc5c(cc4C(=O)N4Cc6ccccc6C[C@H]4CN4CCOCC4)OCO5)n4c3CCCC4)c3ccc(O)cc3)cc2)CC1
InChIKey: MWRSSCMKBFXQIX-KDXMTYKHSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: BCL-2 TR-FRET Assay: The following assay concentrations and times were used: 3 ng BCL-2, 5 μl of 1:100 anti-His Tb-labeled donor, 5 μl of 1:100 Dye-labeled acceptor, 5 μl of 1:40 BCL-2 Peptide Ligand, and 2 ul of test compound, with 60 min incubation time. The results of the assay were read using a Clariostar (BMG Labtech) plate reader with the following parameters: TR FRET, 340ex/620 and 665em; 60 μsec Delay; and 500 μsec integration.
Measurement: IC50 = 7.5 nM (pChEMBL 8.119999885559082)
Descriptors: MW 851.02, ALogP 6.06, HBA 10, HBD 1, PSA 120.26, QED 0.19
Compound (SMILES): N=C/C(C(=O)Nc1cc2c(cc1N1CCOCC1)C(=O)N([C@@H]1CCOC[C@H]1F)C2)=C1/N=CC=CN1
InChIKey: MLIQFBGVLMIWMB-XQIDDSHGSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biological Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 uM ATP; 10 uM MgCl2; 125 uM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 3 mM IRAK1; 62.5 uM ATP; 5 uM MgCl2, and 62.5 uM IRAK1 peptide in reaction buffer for 60 min.Assays of compounds for kinase inhibition were performed using inhibitors serially-diluted in dimethyl sulfoxide, which was accomplished with a LabCyte Echo 555 liquid dispenser. 384 well assay plates spotted with compound received 4 ul of a 2× substrate (ATP+peptide) mix in reaction buffer, followed by 4 ul of 2× enzyme diluted in reaction buffer. Reactions were halted at 60 (IRAK1) or 90 (IRAK4) min by addition of 6 ul of detection buffer, containing EDTA (40 nM final concentration), 0.95 ug of the ADP-binding antibody ADP2, ADP tracer (3 nM final concentration), and 25 uM HEPES. Following a 1 hr incubation, fluorescence polarization of the ADP2-antibody TRACER complex was read on a Tecan M1000 plate reader using a 635/20 excitation filter in combination with a 670/20 emission filter.
Measurement: Ki = 1 nM (pChEMBL 9)
Descriptors: MW 482.52, ALogP 1.59, HBA 8, HBD 3, PSA 119.35, QED 0.43
Compound (SMILES): CC(C(=O)Nc1ccccn1)(c1ccccc1)n1ncc2c1nc(N)n1nc(-c3ccco3)nc21
InChIKey: IXZMDGUWPYXWSC-UHFFFAOYSA-N
Target: Adenosine deaminase (SINGLE PROTEIN), Homo sapiens
UniProt: P00813
Assay: Luciferase Reporter Assay: HEK293-A2AR-luc2p/CRE/Hygro cells were seeded at a density of 5,000 cells/well in DMEM with 1% FBS and 1 U/mL adenosine deaminase (ADA) (Sigma). After 18 h, the cells were treated with 3 nM CGS21680 plus a series dilution of A2AR antagonist, the compounds disclosed herein at the concentration of 0.110000 nM, prepared in DMEM with 1% FBS. After 5 h incubation, the luciferase activity in cells were measured using the Bright-Glo Luciferase Assay System (Promega) according to manufacturer's instructions. The luminescence signal was measured using a PHERAstar FS plate reader (BMG Labtech). Luminescence intensity from 10 μM preladenant treatment was set as 0%. Maximal luminescence intensity was determined in the presence of 3 nM CGS21680 and was set as 100%. The IC50 value was calculated from a dose dependent inhibition curve across the range of compound concentrations.
Measurement: IC50 = 15.3 nM (pChEMBL 7.820000171661377)
Descriptors: MW 465.48, ALogP 3.11, HBA 10, HBD 2, PSA 142.05, QED 0.4
Compound (SMILES): CSSC1CCN(c2nc(C(F)(F)c3ccccc3)nc3c2nnn3Cc2ccccc2Cl)C1
InChIKey: RTKWAUCEOZNURR-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Radioligand Binding Assay: he affinity of the compounds of the invention for cannabinoid receptors was determined using recommended amounts of membrane preparations (PerkinElmer) of human embryonic kidney (HEK) cells expressing the human CNR1 or CNR2 receptors in conjunction with 1.5 or 2.6 nM [3H]-CP-55,940 (Perkin Elmer) as radioligand, respectively. Binding was performed in binding buffer (50 mM Tris, 5 mM MgCl2, 2.5 mM EDTA, and 0.5% (wt/vol) fatty acid free BSA, pH 7.4 for CB1 receptor and 50 mM Tris, 5 mM MgCl2, 2.5 mM EGTA, and 0.1% (wt/vol) fatty acid free BSA, pH 7.4 for CB2 receptor) in a total volume of 0.2 mL for 1 h at 30° C. shaking. The reaction was terminated by rapid filtration through microfiltration plates coated with 0.5% polyethylenimine (UniFilter GF/B filter plate; Packard). Bound radioactivity was analyzed for Ki using nonlinear regression analysis (Activity Base, ID Business Solution, Limited), with the Kd values for [3H]CP55,940 determined from saturation experiments.
Measurement: Ki = 1372 nM (pChEMBL 5.860000133514404)
Descriptors: MW 519.05, ALogP 5.65, HBA 8, HBD 0, PSA 59.73, QED 0.29
Compound (SMILES): CS[C@H]1O[C@@H](c2ccc(C)c(Cc3ccc(CCCCCCC(=O)NC(CO)(CO)CO)cc3)c2)[C@H](O)[C@@H](O)[C@@H]1O
InChIKey: REJWSHCJKXUQDW-IXYVTWBDSA-N
Target: Sodium/glucose cotransporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P13866
Assay: In Vitro Human SGLT1 Inhibition Assay: Human sodium/glucose co-transporter type 1 (SGLT1; accession number NP_000334; GI: 4507031) was cloned into pIRESpuro2 vector for mammalian expression (construct: HA-SGLT1-pIRESpuro2). HEK293 cells were transfected with the human HA-SGLT1-pIRESpuro2 vector and the bulk stable cell line was selected in presence of 0.5 μg/mL of puromycin. Human HA-SGLT1 cells were maintained in DMEM media containing 10% FBS, 1% GPS and 0.5 μg/mL of puromycin. The HEK293 cells expressing the human HA-SGLT1 were seeded in 384 well plates (30,000 cells/well) in DMEM media containing 10% FBS, 1% GPS and 0.5 μg/mL of puromycin, then incubated overnight at 37 C, 5% CO2. Cells were then washed with uptake buffer (140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 5 mM Tris, 1 mg/mL bovine serum albumin (BSA), pH 7.3). Twenty microliters of uptake buffer with or without testing compounds were added to the cells. Then, 20 microliters of uptake buffer containing 14C-AMG (100 nCi) were also added to cells. The cell plates were incubated at 37° C., 5% CO2 for 1-2 hours. After washing the cells with uptake buffer, scintillation fluid was added (40 microliters/well) and 14C-AMG uptake was measured by counting radioactivity using a scintillation coulter (TopCoulter NXT; Packard Instruments).
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 591.77, ALogP 1.75, HBA 9, HBD 7, PSA 159.71, QED 0.15
Compound (SMILES): CN1CCN2CCNc3ccn4ncc(c4n3)-c3cc(ccc3F)OC[C@@H]2C1
InChIKey: DDOQCWDRKQWBQX-AWEZNQCLSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: in vitro peptide-based kinase assay: The inhibition of LRRK2 kinase was assessed using LRRK2 recombinant protein in an in vitro peptide-based kinase assay.ProtocolA radiometric protein kinase assay (33PanQinase® Activity Assay) is used for measuring the kinase activity. All assays are performed in 96-well FlashPlates from Perkin Elmer in a 50 μl reaction volume. The reaction cocktail is pipetted in 4 steps in the following order:    10 μl of non-radioactive ATP solution (in H2O)    25 μl of assay buffer/[γ-33P]-ATP mixture    5 μl of test sample in 10% DMSO    10 μl of enzyme/substrate mixtureThe assay for LRRK2 contains 70 mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 μM Na-orthovanadate, 1.2 mM DTT, 50 μg/ml PEG20000, ATP (0.3 μM), [γ-33P]-ATP (approx. 4×1005 cpm per well), protein kinase LRRK2 (7.3 nM) and substrate (GSK3(14-27), 1.0 μg/50 μl).The kinase is obtained from Invitrogen Corporation.The reaction cocktails were incubated at 30° C. for 60 minutes. The reaction was stopped with 50 μl of 2% (v/v) H3PO4, plates were aspirated and washed two times with 200 μl 0.9% (w/v) NaCl. Incorporation of 33Pi (counting of cpm) was determined with a microplate scintillation counter.CompoundsThe compounds are dissolved to 10 mM in DMSO. Where needed, solutions are sonicated in a bath sonicator.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 382.44, ALogP 1.96, HBA 7, HBD 1, PSA 57.93, QED 0.64
Compound (SMILES): CC(=O)OCC(C)(OC(C)=O)[C@@H]1C[C@H](OC(C)=O)[C@@]2(C)Oc3cc(-c4cccnc4)oc(=O)c3[C@H](O)[C@@H]2C1
InChIKey: NIGGKHQHUNJNPM-IWOUTKOISA-N
Target: Sterol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75908
Assay: Inhibitory Effect on ACAT2 Activity: 1. Purpose:The analogues of Pyripyropene A structure were tested for their inhibitory effect on ACAT2 activity at intact cellular level by a method in which a fluorescent-labeled sterol is used for determining ACAT2 activity.2. Principle:An inhibition curve was plotted based on that different concentrations of compounds would cause the fluorescence intensity to change by inhibiting the synthesis of the ester containing NBD22-fluorescent labeled sterol, thereby IC50 was calculated.3. Process:HepG2 cells were cultured in a 96-well plate at a starting density of 1.5×104 cells per well for 24 hours. After the cholesterol mixture was well mixed, the cells were cultured for another 24 hours. Then NBD22-fluorescent labeled sterol at a final concentration of 0.5 μg/ml, and compounds at a final concentration gradient of 0, 0.008, 0.04, 0.2, 1 and 5 μM were added. Three wells were set for each concentration. After incubating for 6 hours, the fluorescence intensity was measured using a fluorescence analyzer (E488, A535). The fluorescence intensity values were plotted against different concentrations of the compounds and IC50 was obtained.
Measurement: IC50 = 245 nM (pChEMBL 6.610000133514404)
Descriptors: MW 529.54, ALogP 2.73, HBA 11, HBD 1, PSA 151.46, QED 0.43
Compound (SMILES): CS(=O)(=O)c1ccc([C@@H](CO)NC(=O)c2ccc3c(c2)nc(C(F)F)n3Cc2cccc3c2OC(F)(F)O3)cc1
InChIKey: MDVIADAUTJSVKH-LJQANCHMSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay : Compounds described herein were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. #NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. #RPNQ0010) were added to achieve 50 ag of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORγ inverse agonist T0901317 (SigmaAldrich, Cat. #T2320). The percent inhibition vs. concentration data were fit into a four-parameter model, and IC50 values were calculated from the fit as the concentrations corresponding to the inflection points on the dose-response curves.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 579.53, ALogP 4.21, HBA 8, HBD 2, PSA 119.75, QED 0.3
Compound (SMILES): COc1ccc([N+](=O)[O-])c(S(=O)(=O)C(C)C)n1
InChIKey: NUBIOLHAPMEOLX-UHFFFAOYSA-N
Target: Thioredoxin reductase 1, cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: Q16881
Assay: Inhibition of Recombinant TrxR1: Small molecule inhibition of recombinant thioredoxin reductase 1 (TrxR1) and gluthathione reductase (GR) was examined in 96-well plate format. 30 nM TrxR1 was incubated in the presence of 250 μM NADPH, 0.1 mg/ml BSA, and various concentrations of compound (1% DMSO final) in 50 mM Tris (pH 7.5) and 2 mM EDTA buffer for 15 minutes. Following the incubation period, 2 mM DTNB was added to each well and the change in O.D. at 412 nm was followed. Percent activity was determined using DMSO vehicle and no TrxR1 (blank) controls. 2 nM GR was incubated in the presence of 250 μM NADPH, 0.1 mg/ml BSA, and various concentrations of compounds (1% DMSO final) in 50 mM Tris (pH 7.5) and 2 mM EDTA buffer for 15 minutes. Following the incubation period, 1 mM GSSG was added to each well and the change in O.D. at 340 nm was followed. Percent activity was determined using DMSO vehicle and no GR (blank) controls.
Measurement: IC50 = 204 nM (pChEMBL 6.690000057220459)
Descriptors: MW 260.27, ALogP 1.18, HBA 6, HBD 0, PSA 99.4, QED 0.59
Compound (SMILES): COc1cc(-c2cc(C)c(-c3ccc(F)c(CNc4ccc([C@H]5C[C@@H]5C(=O)O)nc4)c3)c(C)c2)ccn1
InChIKey: BWUZIKYFTVCZAH-DQEYMECFSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: IPOne assay: IP1 accumulation measurements using the IPOne assay system1321N1 cells stably expressing human GPR40 receptor (Euroscreen, Belgium) are seeded 24 h before the assay in white 384-well plates in culture medium containing 10% FCS, 1% Na-Pyruvate and 400 μg/mL G418. IP1 is assayed according to the manufacturer's description (Cisbio Bioassays, France). In brief, the assay is started by substitution of the culture medium by stimulation buffer (Hepes 10 mM, CaCl2 1 mM, MgCl2 0.5 mM, KCl 4.2 mM, NaCl 146 mM, glucose 5.5 mM and LiCl 50 mM, pH 7.4). Cells are stimulated for 1 h at 37° C., 5% CO2 by addition of the compounds that are diluted in stimulation buffer containing LiCl. Assays are stopped by adding HTRF-conjugates (IP1-d2 and Anti-IP1 cryptate Tb) and lysis buffer, provided by the manufacturer. After an incubation time of 1 h at room temperature plates are measured using an EnVision, Perkin Elmer. The obtained fluorescence ratios at 665/615 nM are then used to calculate the pEC50 values using Assay Explorer 3.3 Software (Accelrys, Inc.) by interpolation using an IP1 reference curve and subsequent sigmoidal curve fitting allowing for a variable hill slope.
Measurement: EC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 497.57, ALogP 6.38, HBA 5, HBD 2, PSA 84.34, QED 0.29
Compound (SMILES): CC(C)(C)NCCNc1ccc(-c2nnc(C(F)(F)F)o2)nc1C1CCN(c2ncnc3c2C(C)(C)C(=O)N3)CC1
InChIKey: JXSXWPCGHMFVGR-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase alpha-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P51812
Assay: Inhibition of RSK2 (unknown origin)
Measurement: IC50 = 0.16 nM (pChEMBL 9.800000190734863)
Descriptors: MW 573.62, ALogP 4.35, HBA 10, HBD 3, PSA 133.99, QED 0.35
Compound (SMILES): Cc1cc(Nc2ncnc(-c3ccc(O[C@H]4CCN(C(=O)CO)C[C@H]4F)c(C#N)c3)n2)ccc1N1CCN(C2COC2)CC1
InChIKey: SQZMEALFOPIMFM-NAKRPHOHSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biological for hJAK2: Enzymatic activity of hJAK2 was measured using a LANCE homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate (PTK). An increase in the amount of phopshorylated peptide results in an increase in TR-FRET signal. hJAK2 was expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from PerkinElmer. hJAK2 enzyme was assayed under initial rate conditions in the presence of 2×Km ATP (30 μM) and 1 μM peptide, 50 mM Tris-Cl (pH 7.5), 10 mM MgCl2, 1mM dithiothreitol, 0.01% BSA, 0.05% (v/v) DMSO at the following concentration of enzyme: hJAK2 at 0.3 nM. After an assay reaction time of 40 minutes at 25° C., reactions were terminated with EDTA and LANCE Detection Buffer.Amount of phosphorylated peptide was determined by the addition of 20 nM SA-APC and 1 nM europium cryptate labeled anti-phospho PTK monoclonal antibody PY66 and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the doseresponse curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations. These assays generally produced results within 3-fold of the reported mean.
Measurement: IC50 = 64 nM (pChEMBL 7.190000057220459)
Descriptors: MW 602.67, ALogP 2.29, HBA 11, HBD 2, PSA 139.97, QED 0.39
Compound (SMILES): CC(C)CNc1cc(Nc2ncc(C#N)cc2Cl)ncc1C(=O)NC[C@@H](F)C(C)(C)O
InChIKey: KTXPPFHNIAYAML-QGZVFWFLSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 4.3 nM (pChEMBL 8.369999885559082)
Descriptors: MW 448.93, ALogP 3.65, HBA 7, HBD 4, PSA 122.96, QED 0.46
Compound (SMILES): N[C@H](CNC(=O)c1cc2nccc(C(F)(F)F)c2s1)C(=O)O
InChIKey: BVMISDVPLOGTCO-ZCFIWIBFSA-N
Target: Glutamate receptor ionotropic, NMDA 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q05586
Assay: Scintillation Proximity Assay (SPA): To determine the affinity of the compounds of the present invention a SPA is used. The assay is run in a 384-plate format (OptiPlate-384) where each well contains a mix of 5 μL of test compound, 5 μL NR1s1s2 (ligand binding domains of the NMDA receptor, MW 35.6 kDa, 0.075 ug/well final), 5 μL [3H]-MDL-105,519 (radiolabelled, high affinity N-methyl-D-aspartate (NMDA) glutamate receptor antagonist at the glycine site obtained fromSigma Aldrich, final concentration 5 nM, Kd=1.3 nM), 5 μL streptavidin coated imaging beads (Perkin Elmer cat. No.: RPNQ0273, 8 ug/well). The assay buffer contains 100 mM HEPES-NaOH, 150 mM NaCl, 1 mM EDTA, 10% glycerol at pH 7.4 in ultra-pure water. Non-specific binding is defined by inclusion of 10 μM L-689,560 (highly potent NMDA antagonist) and total binding by 1% DMSO. Following 30 minutes incubation in the dark (shaker, Multi-microplate Genie), the SPA beads are allowed to settle for 3 h after which the signal is read on a Viewlux instrument (Perkin Elmer). Normalized data are used to calculate IC50 and Ki values.
Measurement: Ki = 360 nM (pChEMBL 6.440000057220459)
Descriptors: MW 333.29, ALogP 1.46, HBA 5, HBD 3, PSA 105.31, QED 0.79
Compound (SMILES): COc1ccc(C#N)c2c(=O)n(CCOc3ccc(Cl)cc3-c3ccnc4c(C(=O)O)csc34)c(C)nc12
InChIKey: WQWAMNHDCCSIPW-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 = 12.6 nM (pChEMBL 7.900000095367432)
Descriptors: MW 546.99, ALogP 5.29, HBA 9, HBD 1, PSA 127.33, QED 0.29
Compound (SMILES): COC(=O)N1c2ccc3c(nc(NS(=O)(=O)c4ccccc4)n3[C@@H]3CCC[C@@H](C(=O)O)C3)c2CC[C@@H]1C
InChIKey: PPJFPDJMQHNWPW-RCCFBDPRSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: HTRF Biochemical Assay for CBP and BRD4 Activity: The assay was performed in a final volume of 6 μL in assay buffer containing 50 mM Hepes (pH 7.5, (0.5M Hepes, pH 7.5 solution; Teknova H1575)), 0.5 mM GSH, 0.01% BGG (0.22 μM filtered, Sigma, G7516-25G), 0.005% BSA (0.22 μM filtered, EMD Millipore Corporation, 126575) and 0.01% Triton X-100 (Sigma, T9284-10L). Nanoliter quantities of 10-point, 3-fold serial dilution in DMSO were pre-dispensed into 1536 assay plates (Corning, #3724BC) for a final test concentration of 33 μM to 1.7 nM, top to lowest dose, respectively. 3 of 2× Protein and 3 μL of 2× Peptide Ligand were added to assay plates (pre-stamped with compound). Plates were incubated for varying times at room temperature prior to measuring the signal. TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) was measured on the PHERAstar (BMG, equipped with HTRF optic module [337/520/490]) or on the Envision (PerkinElmer, equipped with the TRF Laser unit, TRF dual mirror D400/D505 and emission filters M520 and M495). Data were reported as percent inhibition compared with control wells based on the following equation: % inh=1−((TR-FRET ratio−AveLow)/(AveHigh−AveLow)) where TR-FRET ratio=(Fluorescence at 520 nm/Fluorescence at 490 nm)*10000), AveLow=average TR-FRET ratio of no enzyme control (n=32), and AveHigh=average TR-FRET ratio of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm. For all assay formats data were reported as percent inhibition compared with control wells based on the following equation: % inh=100*((FLU−AveLow)/(AveHigh−AveLow)) where FLU=measured Fluorescence, AveLow=average Fluorescence of no enzyme control (n=32), and AveHigh=average Fluorescence of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 526.62, ALogP 4.56, HBA 7, HBD 2, PSA 130.83, QED 0.5
Compound (SMILES): COc1ccc(S(=O)(=O)c2c(N)[nH]c3c(C4CCCC4)ncnc23)cc1
InChIKey: SJUYXUIQTSTVLZ-UHFFFAOYSA-N
Target: Free fatty acid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O15552
Assay: Ca-Flux Functional Assay: GPR43 agonist/PAM activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by FLIPR (manufactured by Molecular Devices). GPR43 mediated increases in intracellular Ca2+ concentration were readily detected upon activation with sodium acetate. Prior to the assay (24 hours), CHO-K1 Gα16 cells stably expressing human GPR43 were-seeded in cell culture medium in black, clear-bottom 384-well plates (Corning Inc) and grown overnight at 37° C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (Molecular Devices) diluted in HBSS containing 25 mM HEPES, 2.5 mM Probenecid, 0.1% BSA for 1 hour at 37° C., 5% CO2. 10 point half log concentration response curves of sodium acetate from 10 mM were conducted prior to the testing of compounds to calculate the sodium acetate concentration that produces 20% of the maximal response (EC20). Test compounds (at 10 point half log concentration response curves from 10 μM) were added in the presence of sodium acetate to achieve a final concentration that produces approximately 20% maximal response as calculated from the previous experiment. The changes in fluorescent signal were monitored by FLIPR upon addition of the compound/EC20 sodium acetate mix. The EC50 values were determined from ten point concentration response curves. Curves were generated using the average of two wells for each data point.
Measurement: EC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 372.45, ALogP 3.04, HBA 6, HBD 2, PSA 110.96, QED 0.73
Compound (SMILES): Cc1cc(OCCCc2c3n(c4c(-c5c(C)nn(C)c5C)c(Cl)ccc24)[C@H](C)CN(c2cc(C(=O)N4CCOCC4)cc4cc(C(=O)O)n(C)c24)C3=O)cc(C)c1Cl
InChIKey: LJTDOKVBHULNSR-RUZDIDTESA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: TR-FRET Binding Assay: Measurements were carried out in OptiPlate-384 White Opaque 384-well plates (Perkin Elmer. Shelton, Conn.). The assay was run using a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) that was purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification, 300 nM FITC-Bak peptide, 1 nM recombinant Maltose Binding Protein (MBP) tagged Mcl-1 (residues 172-327) and 1 nM terbium conjugated-MBP antibody were added to assay buffer (4.5 mM Monobasic potassium phosphate, 15.5 mM Dibasic potassium phosphate, 1 mM Sodium EDTA, 50 mM NaCl, 1 mM DTT, 0.05% Pluronic F-68, pH 7.5). Compounds are diluted in DMSO in a 16-point, semilog serial dilution scheme. For the BAK FITC TR-FRET assay 50 nL compound is added to 20 μL of assay buffer containing Bak-FITC. MBP tagged Mcl-1 protein and antibody, for a final DMSO concentration of 0.25% and a top concentration of 10 μM. A FITC-Bak peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. The plate was mixed and incubated for 3 hours at room temperature. TR-FRET signal (Delta F) is measured on a Biotek Cytation 3 multimode plate reader equipped with a filter cube containing an Ex 340/30 Em 620/10 nm filter and an Ex 340/30 Em 520 filter.
Measurement: Ki < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 825.79, ALogP 8.48, HBA 9, HBD 1, PSA 124.06, QED 0.15
Compound (SMILES): Cn1cc(-c2cnn(Cc3ccccc3)c2)c(-c2cccs2)cc1=O
InChIKey: QVSBHAPRISVXJW-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: In Vitro CBP Inhibition: The CBP-inhibitory activity of the compounds described herein was determined by calculating the IC50. More specifically, CBP inhibitor activity was assayed as follows: CBP 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. CBP binding and inhibition is 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 CBP (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 min incubation at room temp, Alpha-streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 calculated using a four parameter non-linear curve fit.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 347.44, ALogP 4.03, HBA 5, HBD 0, PSA 39.82, QED 0.56
Compound (SMILES): O=P(O)(O)CS(=O)(=O)C[C@H]1O[C@@H](n2ncc3c(NC4CCCC4)nc(CO)nc32)[C@H](O)[C@@H]1O
InChIKey: PFHXWPOGQPCOBM-LSCFUAHRSA-N
Target: 5'-nucleotidase (SINGLE PROTEIN), Homo sapiens
UniProt: P21589
Assay: Biochemical Assay: Assay Reaction ConditionsAssay Volume: 70 μlReaction Volume: 50 μlCD73: 0.3208 nMAMP: 15 μMAssay Buffer: 2 5 mM Tris-HCL, pH 7.4, 0.01% Brij-35, 0.01% BSA, 5 mM MgCl2Assay Procedure:Used 384 clear plate.Made dose titration of testing compounds in assay buffer, 10 points ½ log titrations in duplicates starting at 100 μM.Added 25 μl of CD73 to each well for a final concentration of 320 pM.Incubated at RT for 15 min.Added 25 μl of AMP to each well for a final concentration of 15 μM.Incubated at RT for 10 min.Added 10 μl of Malachite Green Reagent A, incubate at RT for 10 min.Added 10 μl of Malachite Green Reagent B, incubate at RT for 45 min.Read the Absorbance on Envision plate reader using excitation filter: Cy5 620 nM.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 507.46, ALogP -1.16, HBA 12, HBD 6, PSA 217.22, QED 0.23
Compound (SMILES): COc1ncccc1-c1ccncc1N(CC(F)(F)F)C(=O)c1cc(C(F)(F)F)cc(S(=O)(=O)N(C)C)c1
InChIKey: GVPADOKKFYRODA-UHFFFAOYSA-N
Target: G-protein coupled bile acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDU6
Assay: cAMP Assay: CHO-dhfr(minus) cells expressing human GPBAR1 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), lx HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. The assay was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaked for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH, Hamburg Germany), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The measured signal at 730 nm has to be corrected for the ruthenium background, the direct excitation of Alexa and the buffer control. The FRET signal is calculated as follows: FRET=T730−Alexa730−P(T645−B645) with P=Ru730−B730/Ru645−B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 118 nM (pChEMBL 6.929999828338623)
Descriptors: MW 562.49, ALogP 4.63, HBA 6, HBD 0, PSA 92.7, QED 0.39