lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@]1(C(=O)O)CC[C@H](C(=O)N2CCC3(S(=O)(=O)c4ccc(F)cc4)c4ccc(C(F)(C(F)(F)F)C(F)(F)F)cc4OCC23)CC1
InChIKey: ALHXVPACIBAJJZ-FVSSTLBOSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Mus musculus
UniProt: P51450
Assay: General RORgamma SPA binding assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 79 nM (pChEMBL 7.099999904632568)
Descriptors: MW 669.59, ALogP 6.06, HBA 5, HBD 1, PSA 100.98, QED 0.31
Compound (SMILES): CC(C)(C)c1nc2c(nnn2Cc2ccccc2Cl)c([C@]2(O)C[C@H](C(F)F)C2)n1
InChIKey: YCWCOXJTEIBYMU-NJDVURJYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 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), 1×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. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), 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 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.EC50 values were determined using Activity Base analysis (ID Business Solution, Limited). The EC50 values for a wide range of cannabinoid agonists generated from this assay were in agreement with the values published in the scientific literature.The compounds of the invention are CB2 receptor agonists with EC50 below 1 μM and selectivity versus CB1 in the corresponding assay of at least 10 fold. Particular compound of the invention are CB2 receptor agonists with EC50 below 0.05 μM and selectivity versus CB1 in the corresponding assay of at least 500 fold.
Measurement: EC50 = 9.8 nM (pChEMBL 8.010000228881836)
Descriptors: MW 421.88, ALogP 4.08, HBA 6, HBD 1, PSA 76.72, QED 0.69
Compound (SMILES): O=C(Nc1ccc(F)c(Nc2ncccc2-c2ncnc3[nH]cnc23)c1F)c1ccc(N2CCSCC2)c(C(F)(F)F)c1
InChIKey: RYTCJJYVABAUSL-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Experiment on VEGFR-2 Tyrosine Kinase Inhibitory Activity: The inhibitory activities of the compounds of the present invention against VEGFR-2 tyrosine kinase were analyzed using ADP-Glo kinase assay kit commercially available from Promega. In the principle of the analysis, kinase, a substrate and ATP are reacted with one another, and then ADP-Glo solution is added thereto. The ATP is removed while leaving the produced ADP, and the remaining ADP is converted to ATP by use of a kinase detection reagent, and the ATP is reacted with a luciferin substrate, and the emitted luminescence is measured. Specifically, 5 μl of each compound (5×) was added to each well, and 10 μl of kinase enzyme was added to each well, and then 10 μl of a 1:1 mixture of a kinase substrate and an ATP solution was added to each well (substrate: 5 μg/5 μl; ATP: 20 μM/5 μl). These substances were allowed to react (30° C. and 800 rpm) in a reactor under a light-shielded condition for 30 minutes, and 25 μl of ADP-Glo solution was added to each well and allowed to react under a light-shielded condition for 40 minutes (RT; 150 to 170 rpm). 50 μl of a kinase detection reagent was added to each well and allowed to react under a light-shielded condition for 30 minutes (RT; 150 to 170 rpm), and then the luminescence of each well was measured with a luminometer (Molecular Devices, LMax II 384) and converted to IC50 values.
Measurement: IC50 = 4980 nM (pChEMBL 5.300000190734863)
Descriptors: MW 612.59, ALogP 6.26, HBA 8, HBD 3, PSA 111.72, QED 0.19
Compound (SMILES): COc1cccc(Oc2ccc(-c3nn([C@@H]4CCCNC4)c4c(OC)cncc34)cc2)c1F
InChIKey: XNJQHXBRQQSGDT-QGZVFWFLSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Assay: Measurements were performed in a reaction volume of 15 μL using 384-well assay plates. Kinase enzyme, inhibitor, ATP and 1 μM peptide substrate were incubated in a reaction buffer compose of Hepes 50 mM (pH7.0), NaN3 0.02%, BSA 0.01%, Orthocanadate 0.1 mM. After one hour, the kinase reaction was quenched by the addition of Eμ-labeled antibody and XL-665 in 1ÃDetection buffer containing 60 mM EDTA (Cisbio), and the mixture was allowed to incubate for one hour. The HTRF signal was measured on a multimode plate reader (EnVisionÂ® Multilabel Reader, Perkin Elmer) with an excitation wavelength (λEx) of 330 nm and detection wavelengths (λEm) of 615 and 665 nm. Activity was determined by the ratio of the fluorescence at 665 nm to that at 615 nm. For each compound, enzyme activity as measured at various concentrations of compound, Negative control reactions were performed in the absence of inhibitor in two replicates and eight no enzyme controls.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 448.5, ALogP 4.97, HBA 7, HBD 1, PSA 70.43, QED 0.45
Compound (SMILES): Cc1nnn(Cc2cc(C(F)(F)F)ccc2/C=C/C(=O)N2CCN(Cc3cnn(C)c3)CC2)n1
InChIKey: NHNORVLGDSHGHK-GQCTYLIASA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: ATX Assay: Reagents LPC (oleoyl (18:1)) was purchased from Avanti Polar Lipids (Alabaster, Ala.) and solubilized in methanol to 20 mM. Amplex Red was obtained from Invitrogen Life Technologies (Paisley, UK) and dissolved in DMSO to 10 mM. Choline oxidase and horseradish peroxidase (HRP) were obtained from Sigma Aldrich (Dorset, UK) and dissolved in HBSS to 20 U/ml and 200 U/ml respectively. All reagents were stored at −20° C. in single use aliquots. All experimental measurements were performed in assay buffer made up immediately prior to use (HBSS, 0.01% BSA essentially fatty acid free).Protein Recombinant human ATX was prepared at Novartis (Basel, CH) in a human embryonic kidney (HEK) cell preparation, and stored in single use aliquots of 26 mg/ml (26 μM) stocks stored at −80° C.Method All experimental measurements were performed in black 384 well polystyrene (low volume, round bottom, Corning (3676)) plates. PerkinElmer EnVision (Fluorescence Intensity/Absorbance Monochromator) or Tecan Infinite 200 PRO series plate reader was used to detect change in fluorescent intensity.Assessing ATX inhibition ATX activity was determined by measurement of released choline in reactions containing ATX (10 nM), choline oxidase (0.1 U/ml), HRP (100 U/ml), amplex red (50 μM) and LPC 18:1 (10 μM). Compounds of the invention were prepared as 10 point serial dilutions from 1 μM in duplicate and pre-incubated with ATX at 37° C. for 20 minutes prior to the addition of remaining reagents. The liberated choline was measured from changes in fluorescence intensity (λex 530 nm, λem 590 nm) of the product resurofin at 37° C. every 2 minutes over a 40-minute period. ATX activity was measured as a slope of the linear portion of the progress curve, typically between 14 to 24 minutes.Data analysis Slope data was exported to Graphpad prism (Graphpad software, San Diego, Calif.)
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 474.49, ALogP 2.14, HBA 8, HBD 0, PSA 84.97, QED 0.51
Compound (SMILES): CCn1cc(Nc2ncc(C(N)=O)c(NCc3c(F)cccc3C(F)(F)F)n2)cn1
InChIKey: GLXJKUDCHGMWME-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Kinase Activity Assay: JAK1 (the final concentration was 10 nM); ATP (the final concentration was 10 μM); ULight-labeled JAK-1 (Tyr1023) Peptide (the final concentration was 100 nM); the enzymatic reaction time was 2 hours. The maximum final concentration of compounds was 2.5 μM. After a 3-fold gradient dilution, 11 concentrations were obtained, with the minimum final concentration of 0.042 nM. The final DMSO concentration was 1%.
Measurement: IC50 = 29.3 nM (pChEMBL 7.53000020980835)
Descriptors: MW 423.37, ALogP 3.31, HBA 7, HBD 3, PSA 110.75, QED 0.5
Compound (SMILES): Cc1c(-c2ccc(C(N)=O)c3[nH]c(C4=CCOCC4)cc23)cccc1-n1cnc2ccc(F)cc2c1=O
InChIKey: QVKADZBHALZKOL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) : The in-house BTK corresponds to recombinant human catalytic domain (aa 393-659), which was expressed in SF9 cells with an N-terminal his tag and purified by immobilized metal affinity chromatography. BTK was mixed with peptide substrate (biotin-TYR1, Sequence: Biotin-(Ahx)-GAEEEIYAAFFA-COOH, 0.2 μM final) at varying inhibitor concentrations in reaction buffer: 50 mM MOPSO pH 6.5, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTT, 0.01% BSA, 0.1 mM Na3VO4 and 0.001 mM ATP. After about 60 mM incubation at room temperature, the reaction was quenched by addition of EDTA (final concentration: 100 mM) and developed by addition of detection reagents (final approximate concentrations: 30 mM HEPES pH 7.0, 0.06% BSA, 0.006% Tween-20, 0.24 M KF, 80 ng/mL PT66K (europium labeled anti-phosphotyrosine antibody cat #61T66KLB Cisbio, Bedford, Mass.) and 0.6 μg/mL SAXL (Phycolink streptavidin-allophycocyanin acceptor, cat #PJ25S, Prozyme, San Leandro, Calif.). The developed reaction was incubated in the dark for about 60 mM at room temperature, then read via a time-resolved fluorescence detector (Rubystar, BMG) using a 337 nm laser for excitation and monitoring emission wavelength at 665 nm. Within the linear range of the assay, the observed signal at 665 nm was directly related to phosphorylated product and can be used to calculate the IC50 values.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 494.53, ALogP 4.88, HBA 5, HBD 2, PSA 103, QED 0.37
Compound (SMILES): C[C@]1(C(=O)N2CCC3(S(=O)(=O)c4ccc(Cl)cc4)c4ccc(C(F)(C(F)(F)F)C(F)(F)F)cc4CC23)CC[C@H](C(=O)O)CC1
InChIKey: QEFRQORVGXHVSQ-FVSSTLBOSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Mus musculus
UniProt: P51450
Assay: General RORgamma SPA binding assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 670.04, ALogP 6.74, HBA 4, HBD 1, PSA 91.75, QED 0.36
Compound (SMILES): CCS(=O)(=O)c1ccc(Cl)cc1Cn1c(=O)[nH]c2c(Cl)c(CN3CCC[C@@H](CNC(=O)OC(C)(C)C)C3)c(C(F)(F)F)cc2c1=O
InChIKey: RIVWFJKUUKGQSC-KRWDZBQOSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: Binding Activity: DDR1 binding activity was measured using the LanthaScreen(Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). The test compound and the Alexa Fluor® 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen(Registered trademark) Eu-anti-GST antibody. After one-hour reaction at room temperature, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 310 nM (pChEMBL 6.510000228881836)
Descriptors: MW 707.6, ALogP 5.59, HBA 8, HBD 2, PSA 130.57, QED 0.31
Compound (SMILES): O=C(Cc1ccc(-c2cnc3[nH]c(C(=O)O)cc3c2)cc1F)Nc1cc(C2(C(F)(F)F)CC2)on1
InChIKey: IZBLDPHONJVQKO-UHFFFAOYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Competition Binding Assay: Competition binding assays used herein were developed, validated and performed as described in Fabian et al., Nature Biotechnology 2005, 23, 329-336. Kinases were produced as fusions to T7 phage (See, Fabian et al. or WO04/015142) or alternatively, the kinases were expressed in HEK-293 cells and subsequently tagged with DNA for PCR detection (See, WO08/005,310). For the binding assays, streptavidin-coated magnetic beads were treated with biotinylated affinity ligands for 30 min at room temperature to generate affinity resins. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinase, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100× stocks in DMSO and diluted into the aqueous environment. Kds were determined using an eleven point threefold serial dilutions. DMSO or control compounds were was added to control assays lacking a test compound.
Measurement: Kd < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 488.4, ALogP 4.83, HBA 5, HBD 3, PSA 121.11, QED 0.33
Compound (SMILES): C[C@@H]1[C@H](NC(=O)N(C)C)CCCN1c1cnc(C(N)=O)c(Nc2ccc(C3CCN(C4CCC4)CC3)cc2)n1
InChIKey: JTUXDMHTCPSLEV-NTKDMRAZSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk In Vitro Inhibitory Assay: Human Btk kinase (Genbank accession # NP_000052) was purified from insect cells as a full-length construct containing a N-terminal 6×-His tag. Btk kinase activity was determined using a radiometric filter binding assay. Measurements are performed in a low μL reaction volume 384-well assay plates. BTK enzyme (8 nM final in reaction), inhibitor (at requested doses), and 0.2 mg/mL peptide substrate (Poly-Glu-Tyr, 4:1 ratio) are incubated in a reaction buffer composed of 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO for 15 min. followed by addition of 1 μM ATP to start the assay. Kinase reactions are carried out for 120 min. at room temperature. The reaction was stopped by spotting of reaction sample onto P81 cationic exchange paper (Whatman). Unbound phosphate was removed by extensive washing of filters in 0.75% Phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme (via addition of saturating EDTA), kinase activity data for each dose of compound tested was expressed as the percent of remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 534.71, ALogP 3.68, HBA 7, HBD 3, PSA 119.72, QED 0.5
Compound (SMILES): CCS(=O)(=O)c1ccc(Cl)cc1CNC(=O)c1cc(OC(F)(F)F)cc(N2CCN(CC(F)(F)F)CC2)c1
InChIKey: PSZKPIDNIIPBSX-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: LanthaScreen Eu Kinase Binding Assay: DDR1 binding activity was measured by the LanthaScreen (Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). A test compound and the Alexa Fluor (Registered trademark) 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen (Registered trademark) Eu-anti-GST antibody. After reacting at room temperature for one hour, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 85 nM (pChEMBL 7.070000171661377)
Descriptors: MW 587.97, ALogP 4.65, HBA 6, HBD 1, PSA 78.95, QED 0.45
Compound (SMILES): Nc1cc(Cc2cccc(Cc3ccccc3)c2)c2nn[nH]c2n1
InChIKey: QAYFAZVYHUBONM-UHFFFAOYSA-N
Target: Myeloperoxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P05164
Assay: MPO Chlorination Assay (APF Assay): MPO chlorination activity was measured in 100 mM KPi (pH 7.4) by utilizing the non-fluorescent reagent Aminophenyl fluorescein (APF, Invitrogen catalog #A36003). APF is cleaved by (OCl) to yield the fluorescent compound fluorescein. Reactions were carried out in 50 μL total volume by adding a 25 μL mixture of 200 pM myeloperoxidase and 120 mM NaCl to 100 nL inhibitor in 100% DMSO in a 384 well, non-binding surface clear bottom plate (CORNING® #3655). Enzyme, inhibitor, and chloride were preincubated for ten minutes at room temperature.After the ten minute preincubation, 25 μL of an APF mixture containing 10 mM APF, 120 mM NaCl and 10 μM H2O2 was added to the plate using the internal dispensing system of a Hammatsu FDSS 6000. Kinetic determinations were carried out immediately on the FDSS 6000 (3 minute kinetic read, 1 read every second, ex: 485 nm, em: 535 nm). IC50 values for inhibitors were calculated by taking the slope of the linear portion of the kinetic measurement (20 seconds to 80-120 secs).IC50 values were calculated by determining the slope of the linear portion of the kinetic trace (180-540 secs), and using that calculated slope to determine % inhibition occurring at each concentration of inhibitor using the following equation:Y = A + B - A 1 + ( C / x ) D where A=minimal Y value (activity level of inhibited sample), B=maximal Y value (activity level of uninhibited sample), C=Log IC50, D=Hill Slope, x=concentration of inhibitor.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 315.38, ALogP 3.12, HBA 4, HBD 2, PSA 80.48, QED 0.61
Compound (SMILES): CCS(=O)(=O)c1ccc(CNC(=O)c2ccc3c(c2)C(=O)N(C[C@H]2CC[C@H](C(F)(F)F)CC2)[C@H]3C(C)C)cc1
InChIKey: JYQPGLAIMDORIL-YBXDKENTSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay: Compounds of the present invention 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 μg 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 564.67, ALogP 5.93, HBA 4, HBD 1, PSA 83.55, QED 0.43
Compound (SMILES): CCN1CC(C2CCN(CCC(C)C)CC2)OC2(CC2)C1
InChIKey: SYRMTRJVSFHGGC-UHFFFAOYSA-N
Target: Sigma non-opioid intracellular receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99720
Assay: Receptor Radioligand Assay: To investigate binding properties of test compounds to human σ1 receptor, transfected HEK-293 membranes and [3H](+)-pentazocine (Perkin Elmer, NET-1056), as the radioligand, were used. The assay was carried out with 7 μg of membrane suspension, 5 nM of [3H](+)-pentazocine in either absence or presence of either buffer or 10 μM Haloperidol for total and non-specific binding, respectively. Binding buffer contained Tris-HCl 50 mM at pH 8. Plates were incubated at 37° C. for 120 minutes. After the incubation period, the reaction mix was then transferred to MultiScreen HTS, FC plates (Millipore), filtered and plates were washed 3 times with ice-cold 10 mM Tris-HCL (pH7.4). Filters were dried and counted at approximately 40% efficiency in a MicroBeta scintillation counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail
Measurement: Ki < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 294.48, ALogP 3, HBA 3, HBD 0, PSA 15.71, QED 0.78
Compound (SMILES): CC(C)(O)[C@H](F)CNC(=O)c1cnc(Nc2ccc3ncccc3c2)cc1NC1CC1
InChIKey: XOFMANKGLJQULO-HXUWFJFHSA-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% Brij35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction 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 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 423.49, ALogP 3.79, HBA 6, HBD 4, PSA 99.17, QED 0.44
Compound (SMILES): COc1ccc(-c2cc3c(N4CC(C(=O)NCc5ccc(C)cc5)C4)ncnn3c2)cc1F
InChIKey: QYQPRGLOPFEJLB-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kit Wild Type Assay: In each well of a 384-well plate, 0.2 ng/ul final (2 nM) of wild type Kit (Carna Bioscience 08-156) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM Srctide (5-FAM-GEEPLYWSFPAKKK-NH2) and 400 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35s).
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 445.5, ALogP 3.61, HBA 6, HBD 1, PSA 71.76, QED 0.49
Compound (SMILES): CCS(=O)(=O)Nc1nc(Oc2c(C)cccc2C)nc(-c2cc(C)c(=O)n(C)c2)n1
InChIKey: YJFAILHFRJIGGQ-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: In Vitro Enzyme Inhibition Assay-CREBBP Inhibition: Determination of the IC50 for the CREBBP inhibitors disclosed herein was performed as follows: CREBBP was cloned and expressed in E. Coli as His-tag protein and purified by Nickel affinity and gel-filtration chromatography. The protein was further characterized as a single band with the correct molecular weight by SDS-PAGE. CREBBP binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Perkin Elmer). In a 384-well ProxiPlate CREBBP (50 nM final) was combined with peptide (20 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20-minute incubation at room temperature, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After two hours of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit.The ability of the compounds disclosed herein to inhibit CBP activity was quantified and the respective IC50 value was determined.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 429.5, ALogP 2.72, HBA 8, HBD 1, PSA 116.07, QED 0.64
Compound (SMILES): CC(C)c1cccc(-c2cc(COC(C)(C)C(=O)O)nn2Cc2ccccc2Cl)c1
InChIKey: PBSULNYVRQWPIF-UHFFFAOYSA-N
Target: Monocarboxylate transporter 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O15427
Assay: Assay 3: MCT4-Mediated Lactate Transport in MDA-MB-231 Breast Cancer Cells: MDA-MB-231 breast cancer cells may be used to measure MCT4 activity in cells with high native levels of MCT4 and low levels of MCT1 and are known to those with skill in the art. Preparation of BCECF-loaded cells and lactate transport activity may be determined as described for Assay 1.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 426.94, ALogP 5.76, HBA 4, HBD 1, PSA 64.35, QED 0.5
Compound (SMILES): CC(O)c1cccc(-c2cnc(N3CC4(CC4)c4ccc(C(=O)N(C)CCO)cc43)nc2)c1
InChIKey: PHQVEOXQPINGTK-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: cAMP HTRF Assay to Determine the Activity of hPDE4B1: The inhibiting effect of the compounds on the enzyme activity of human PDE4B1 was measured by the quantification of 5′-adenosine monophosphate (5′-AMP), which is formed from 3′,5′-cyclic adenosine monophosphate (cAMP). Human recombinant enzyme, expressed in Sf9 cells, and the HTRF (homogeneous time-resolved fluorescence) detection method were used in the assay.The test compound or water (control) was mixed with the human recombinant PDE4B1 enzyme (4.8 U) in a buffer consisting of 44.4 mM tris-HCl, 5.28 mM MgCl2, 2.64 mM DTT and 0.044% Tween 20 (pH 7.8). After adding the cAMP enzyme substrate (final concentration 40 nM), the mixture was incubated for 30 minutes at room temperature. Then a fluorescence acceptor (Dye2 marked with cAMP), a fluorescence donor (anti-cAMP antibody marked with a europium cryptate) and the non-specific phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine; final concentration 1 mM) were added. After 60 min the fluorescence transfer, which correlates with the amount of remaining cAMP, was measured with a microplate reader (Rubystar, BMG) at λex=337 nm, λem=620 nm and λem=665 nm. The enzyme activity was calculated from the quotient formed from the measured signal at 665 nm and that at 620 nm. The result was expressed as the percentage inhibition of enzyme activity of the control (without PDE4 inhibitor). The enzyme was omitted for measurement of the basal control.[N. Saldou et al., Comparison of recombinant human PDE4 isoforms: interaction with substrate and inhibitors, Cell. Signal. Vol. 10, No. 6, 427-440, 1998].
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 444.54, ALogP 3.44, HBA 6, HBD 2, PSA 89.79, QED 0.61
Compound (SMILES): COc1ccc(-c2[nH]c3ccc(-c4nnc(CC(F)(F)F)o4)cc3c2C(C)C)cc1OC
InChIKey: GGYRRKRXWHYDBA-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 = 1035 nM (pChEMBL 5.989999771118164)
Descriptors: MW 445.44, ALogP 6.13, HBA 5, HBD 1, PSA 73.17, QED 0.39
Compound (SMILES): CC(=O)N(C)C(C)c1c(F)c(Cl)c(-c2ccc3nc(NC(=O)[C@@H]4C[C@@H]4F)cn3c2)c2cn[nH]c12
InChIKey: KNUQDTUWQGZIAY-AFGDWVFNSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: ADP-Glo kinase assay: HPK1 kinase activity was measured by Promega's ADP-Glo kinase assay. In this assay, 5 ng of recombinant human HPK1 (signalchem) is incubated with 5 μL of compounds (0.5% DMSO), 5 μL of MBP (0.5 μg/μl) and 5 μL of ATP (25 μM) in buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg/ml BSA; 5 μM DTT). The assay was started by incubating the reaction mixture in a 96-well plate at 30′ C for 40 minutes. After the incubation, 25 uL ADP-Glo reagent was added and the reaction was incubated at room temperature for 40-min to stop the reaction and degrade residual ATP. The ADP product was then converted to ATP by adding 50 uL per well of detection reagent. Luminescence was detected after 30-min room temperature incubation with the Molecular device I3X plate reader. The IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentration using software routines as implemented in the GraphPad Prism 7 software and SigmaPlot13.0.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 486.91, ALogP 4.51, HBA 5, HBD 2, PSA 95.39, QED 0.43
Compound (SMILES): CC(C)OCCC[C@@H]1CCc2cc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: HPTLZZJKESBNFC-VOQZNFBZSA-N
Target: Sphingosine-1-phosphate lyase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95470
Assay: S1P1 Binding Assay: Membranes were prepared from CHO cells expressing human S1P1. Cells pellets (1×109 cells/pellet) were suspended in buffer containing 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5, 50 mM NaCl, 2 mM EDTA (Ethylenediaminetetraacetic acid) and Protease Inhibitor cocktail (Roche), and disrupted on ice using the Polytron homogenizer. The homogenate was centrifuged at 20,000 rpm (48,000 g) and the supernatant was discarded. The membrane pellets were resuspended in buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 2 mM EDTA and stored in aliquots at −80° C. after protein concentration determination. Membranes (2 μg/well) and 0.03 nM final concentration of 33P-S1P ligand (1 mCi/ml, Perkin elmer or American Radiolabeled Chemicals) diluted in assay buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid free BSA (bovine serum albumin), 1 mM NaF) were added to the compound plates (384 Falcon v-bottom plate (0.5 μl/well in a 11 point, 3-fold dilution). Binding was performed for 45 minutes at room temperature, terminated by collecting the membranes onto 384-well Millipore FB filter plates, and radioactivity was measured by TOPCOUNT®. The competition data of the test compounds over a range of concentrations was plotted as percentage inhibition of radioligand specific binding. The IC50 is defined as the concentration of competing ligand needed to reduce specific binding by 50%.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 425.51, ALogP 4.07, HBA 4, HBD 3, PSA 102.01, QED 0.41
Compound (SMILES): C=CC(=O)N1CCC[C@@H](n2nc(-c3ccc(Oc4cccc(OC)c4Cl)cc3)c3cncc(OCCC)c32)C1
InChIKey: CJPQSTMRNJPTDW-OAQYLSRUSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinases Assay : Btk kinase activity was determined using a homogenous time resolved fluorescence (HTRF) methodology. Measurements were performed in a reaction volume of 15 μL using 384-well assay plates. Kinase enzyme, inhibitor, ATP and 1 μM peptide substrate were incubated in a reaction buffer compose of Hepes50 mM (pH7.0), NaN3 0.02%, BSA 0.01%, Orthocanadate 0.1 mM. After one hour, the kinase reaction was quenched by the addition of E t-labeled antibody and XL-665 in 1×Detection buffer containing 60 mM EDTA (Cisbio), and the mixture was allowed to incubate for one hour. The HTRF signal was measured on a multimode plate reader (EnVision Multilabel Reader, Perkin Elmer) with an excitation wavelength (λEx) of 330 nm and detection wavelengths (λEm) of 615 and 665 nm. Activity was determined by the ratio of the fluorescence at 665 nm to that at 615 nm. For each compound, enzyme activity as measured at various concentrations of compound, Negative control reactions were performed in the absence of inhibitor in two replicates and eight no enzyme controls were used to determine baseline fluorescence levels. IC50s were obtained according to the equation:Y=100/(1+10{circumflex over ( )}((Log IC50−X)*HillSlope)).For BTK assay, [ATP]=80 μM, BTK=3.4 nM.For LYN assay, [ATP]=20 μM, LYN=0.12 n M. For LCK assay, [ATP]=20 μM, LCK=0.2 nM. For BLK assay, [ATP]=20 μM, BLK=0.6 n M.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 547.06, ALogP 6.69, HBA 7, HBD 0, PSA 78.71, QED 0.22
Compound (SMILES): CN(C)c1ccc(-c2cnc3[nH]cc(-c4ccc(N(C)C)nc4)c3c2)cn1
InChIKey: REVMLCZGWSFWGN-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16584
Assay: Radiometric Filter Plate Assay: 200 ng (130 nM) MLK3 (Dundee, DU8313) was incubated with 1 μM inactive MKK7b (Dundee, DU703) in the presence of 2 μM cold ATP (Km) and 0.5 μCi/assay 33P ATP and appropriate concentrations of compounds. After a twenty minute incubation, the reactions were washed through filter plates and read on a scintillation counter.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 358.45, ALogP 3.82, HBA 5, HBD 1, PSA 60.94, QED 0.6
Compound (SMILES): CCNC(=O)C[C@@H]1N=C(c2ccc(Cl)cc2)c2c(cnn2C)-c2cn(C)c(=O)cc21
InChIKey: CIJBRUHXNFAEHT-SFHVURJKSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaLISA Assay: Compounds were diluted by step-down dilution method (final concentration of DMSO was 1%) and added to the wells of a 384 well opti plate at desired concentrations. 5 nM BDR4-BD1 enzyme (produced in-house) and 12 nM of biotinylated substrate were added to the wells, covered and incubated at room temperature (RT) for 1 h. At the end of 1 h 250 ng of GSH acceptor beads were added to the well and incubated for 1 h at RT; then 500 ng of streptavidin donor beads were added and incubated again for 1 h at RT. Plates were read in a Pherastar reader at 680 nm excitation and 570 nm emission. As detailed above, compounds were tested for both BRD4 enzyme inhibitory activities and IC50 were determined.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 423.9, ALogP 2.86, HBA 6, HBD 1, PSA 81.28, QED 0.7
Compound (SMILES): CCOC(=O)C1(c2cn(-c3ccccn3)cn2)CC1CCCN(C(=O)OC(C)(C)C)C(=O)OC(C)(C)C
InChIKey: IUDKZRZTRYNEKG-UHFFFAOYSA-N
Target: Carboxypeptidase B2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96IY4
Assay: Enzyme Inhibitory Assay: HEPES buffered saline (20 mM HEPES, 150 mM NaCl, pH 7.4; hereinafter, referred to as HBS) was used in the preparation of a reaction solution. To 12 μL of a 250 μg/mL TAFI solution, 30 μL of an HBS solution containing 4 U/mL human thrombin, 12 U/mL rabbit lung thrombomodulin, and 12 mM CaCl2 was added, and the mixture was gently stirred. Then, TAFI was activated at room temperature. Ten minutes later, thrombin was neutralized by the addition of 10 μL of 100 μM PPACK (thrombin inhibitor) to terminate the activation of TAFI. The formed TAFIa was stored in ice and diluted immediately before use in determination with 2050 μL of an HBS solution containing BSA (bovine serum albumin) adjusted to 0.1% in terms of the final concentration. A test substance was dissolved in HBS to prepare a 10-fold dilution series of evaluation concentrations. 80 μL of the TAFIa solution and 10 μL of the test substance were added to each well of a 96-well plate and mixed by shaking for 10 minutes. 10 μL of furylacryloyl-alanyl-lysine (FAAK) adjusted to 5 mg/mL was added to each well, and the change in the absorbance of this mixed solution at 330 nm was read for 30 minutes to determine the degradation rate of the substrate.
Measurement: IC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 514.62, ALogP 5.04, HBA 9, HBD 0, PSA 112.85, QED 0.36
Compound (SMILES): Cc1cccc2nc(C(C)Nc3nc(N)nc4[nH]cnc34)c(-c3cccc(F)c3)c(=O)n12
InChIKey: UUVVRXDSNWZLHT-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI3Kdelta Scintillation Proximity Assay A3: The kinase reaction was conducted in polystyrene 384-well matrix white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 0.5%. The PI3K assays were carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 20 μM ATP, 0.2 μCi [γ-33P] ATP, 4 nM PI3Kδ. Reactions were incubated for 210 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 150 mM potassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400 μM ATP. The final concentration of SPA beads was 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1800 rpm for 10 minutes, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent control activity versus the log of the inhibitor concentration using the GraphPad Prism 3.0 software.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 430.45, ALogP 3.23, HBA 8, HBD 3, PSA 126.88, QED 0.4
Compound (SMILES): C=CC(=O)N[C@H]1CCC[C@H]1NC(=O)c1sc2nccc3c2c1NC(=O)N3c1ccc(Nc2ccccn2)nc1C
InChIKey: YKAMPMCYZXYQSV-DLBZAZTESA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan).
Measurement: IC50 = 22.9 nM (pChEMBL 7.639999866485596)
Descriptors: MW 554.64, ALogP 4.78, HBA 8, HBD 4, PSA 141.24, QED 0.24
Compound (SMILES): CC(C)(O)c1ncc(-c2ccc3nc4c(n3c2)N(c2ccccc2OC(F)(F)F)CCC4)cn1
InChIKey: BXEVCQBRIJKBGK-UHFFFAOYSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: TNF or CD40L-Induced HEK-Blue Assay: Test compounds serially diluted in DMSO were plated in an assay plate (Labcyte, Cat. #LP-0200) at final concentrations ranging from 0.004 μM to 25 μM. TNFα (final concentration 0.5 ng/ml) or CD40L (final concentration 30 ng/ml) in assay buffer [DMEM, 4.5 g/l glucose (Gibco, Cat. 21063-029), 10% FBS (Sigma, F4135), 1% Penicillin-Streptomycin (Gibco, Cat. 15140-122), 1% Anti-Anti (Gibco, Cat. 15240-112) and 2 mM L-glutamine (Gibco, Cat. 25030-081)] was then added to the assay plate. After a 30 minute pre-incubation at 37° C. and 5% CO2, HEK-Blue-CD40L cells (InvivoGen, Cat. Code hkb-cd40) containing a NF-κB-driven secreted alkaline phosphatase reporter gene were seeded into the assay plate at a density of 20,000 cells per well. This plate was then incubated for 18 h at 37° C. and 5% CO2. Secreted alkaline phosphatase expression was measured using QUANTI-Blue (InvivoGen, Cat. Code rep-qbl) according to manufacturer's specifications and the assay plate was read on a PerkinElmer Envision at 620 nm. Inhibition data for the test compound over a range of concentrations was plotted as percentage inhibition of the test compound (100%=maximum inhibition). IC50 values were determined after correcting for background [(sample read−mean of low control)/(mean of high control−mean of low control)] where by the low control is DMSO without stimulation and high control is DMSO with stimulation. The IC50 is defined as the concentration of test compound which produces 50% inhibition and was quantified using the 4 parameter logistic equation to fit the data.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 469.47, ALogP 5, HBA 7, HBD 1, PSA 75.78, QED 0.45
Compound (SMILES): Cc1ccc(OCC(F)(F)F)c(C(=O)Nc2cc(-c3nnc(-c4ccco4)o3)ccc2F)c1
InChIKey: SCVQEPXSCKSYLR-UHFFFAOYSA-N
Target: Lysocardiolipin acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q6UWP7
Assay: In Vitro Acyltransferase Assays: ALCAT1 enzymatic activity was determined by measuring the conversion of monolysocardiolipin (MLCL) to cardiolipin or lysophosphatidylglycerol (LPG) to phosphatidylglycerol (PG) in an enzymatic reaction mixture that contained 50 mm Tris/HCl, pH 7.0, 100 μm lysophospholipids, 25 μm [14C]acyl-CoA (50 mCi/mmol, American Radiolabeled Chemicals, Inc), and membrane fraction (0.5-2.5 μg) in a total volume of 200 μl. The reaction was incubated at room temperature for 30 min. The lipids were extracted will chloroform, dried, and separated by thin layer chromatography (TLC) with chloroform:hexane:methanol:acetic acid (50:30:10:5, v/v) or chloroform:methanol:water (65:25:4, v/v). After separation, TLC plates were exposed to a Phosphorlmager screen to visualize the radiolabeled products with a Molecular Dynamics Typhoon Scanner (Sunnyvale, Calif.). In some experiments, NBD-CoA was used at 100 μM to replace the [14C]acyl-CoA in the enzymatic reaction. All quantitative data were expressed as mean±S.E. Statistical analyses for differences between two groups were carried out using a Student's t test.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 461.37, ALogP 5.64, HBA 6, HBD 1, PSA 90.39, QED 0.37
Compound (SMILES): NC(=O)c1c(-c2cccc(F)c2)nn2c1CN(C(=O)NC1CCC(F)(F)CC1)CC2
InChIKey: GXMRXSYTMBWDNW-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 μl prepared from 15 μl 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 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 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 μM ATP, 1.5 μM 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 = 1250 nM (pChEMBL 5.900000095367432)
Descriptors: MW 421.42, ALogP 2.89, HBA 4, HBD 2, PSA 93.25, QED 0.8
Compound (SMILES): CC(C)CN1C(C)Cc2c(ccc3[nH]ncc23)C1c1ccc(/C=C/C(=O)O)cc1
InChIKey: CZDSGCCSLPSVEF-IZZDOVSWSA-N
Target: G-protein coupled estrogen receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99527
Assay: Biological Assay: The compounds of the present invention have an inhibition effect on the binding of E (estrogen) to ER (estrogen receptor), thereby blocking the binding of a complex of E and ER to ERE (estrogen responsive element), and subsequently blocking the expression of downstream luciferase protein.
Measurement: IC50 = 0.64 nM (pChEMBL 9.1899995803833)
Descriptors: MW 389.5, ALogP 4.65, HBA 3, HBD 2, PSA 69.22, QED 0.62