lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COCCNc1cc(NC(=O)N(C)c2nccc(C=O)n2)ncc1C#N
InChIKey: MUFWCTRKSPPARR-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Inhibition Assay: FGFR4:The in vitro activity of FGFR4 was determined by assaying the phosphorylation level of the substrate in the kinase reaction, by means of an HTRF kinase assay kit. The reaction buffer comprised the following components: 5-fold diluted Enzymatic buffer/kinase 5×(main ingredient: 50 mM HEPES, pH 7.0), 5 mM MgCl2, 1 mM DTT; the human recombinant FGFR4 catalytic structural domain protein (amino acids 460-802), diluted with the reaction buffer to a 0.5 ng/μL kinase solution; the substrate reaction solution comprised a biotin labeled tyrosine kinase substrate diluted with the reaction buffer to 500 nM , and 90 μM ATP, and the assay solution comprised an Eu3+ labeled cage-shaped antibody diluted with the assay buffer to 0.125 ng/μL, and 31.25 nM streptavidin labeled XL665).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 355.36, ALogP 1.28, HBA 8, HBD 2, PSA 133.13, QED 0.56
Compound (SMILES): CCN1CCc2cc(OC)c(Nc3ncc(Cl)c(Nc4ccccc4P(C)(C)=O)n3)cc2C1
InChIKey: HUWJQHMDHUJPMQ-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Enzymatic Activity Assay: The enzymatic activity of Wild Type HPK1 (MAP4K1) Kinase was measured in the presence or absence of an inhibitor compound of the present disclosure by preparation of a reaction mixture containing a fluorescein tagged peptide (2 micromolar final concentration), ATP (Adenosine Triphosphate, 500 micromolar final concentration) and Wild Type kinase (6 nM final concentration) in a buffer system comprising 50 mM HEPES (Buffer), 3 mM MgCl2, 5% w/v Trehalose, 0.01% Brij 35 (Detergent), 0.1% Pluronic F127 (Surfactant), 0.1 mM EDTA (Ethylenediaminetetraacetic acid), 0.1 mM EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) and 1 mM DTT (Dithiothreitol) and at a pH of 7.4. The reactions were quenched by the addition of EDTA to a final concentration of 25 mM. A Caliper EZ reader was used to measure the extent of substrate to product conversion.The peptide substrate used in the HPK1 assay was designed based on the amino acid sequence surrounding serine residue 376 of SLP76-Lymphocyte cytosolic protein 2 (a known intracellular substrate of HPK1). This sequence SSFPQSAsLPPYFSQ was modified as follows for use in Caliper assays of HPK1: the serine residues on either side of serine 376 were replaced with alanine, three lysine residues were added at the N-terminus and a fluorescein tag (FAM) was attached to the C-terminus to produce the sequence, FAM-FPQAAsLPPYFAKKK.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 485.96, ALogP 5.25, HBA 7, HBD 2, PSA 79.38, QED 0.44
Compound (SMILES): O=C(Nc1cn2nc(-c3c(-c4ccc(F)cc4)nc4n3CCOC4)ccc2n1)c1cccc(-c2cccnc2)c1
InChIKey: WRVSBUUZEXKWLX-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.
Measurement: IC50 = 4.6 nM (pChEMBL 8.34000015258789)
Descriptors: MW 531.55, ALogP 5.24, HBA 8, HBD 1, PSA 99.23, QED 0.33
Compound (SMILES): COC1CCC([C@@H]2CCN(C(=O)[C@H]3CC[C@H]([C@H](N)CF)CC3)[C@@H]2C(=O)Nc2ccc3oc(C(=O)O)c(Cl)c3c2)CC1
InChIKey: LFHMQEHQWXCRSN-GEOZEOAGSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: 0.35 μg/mL human FXIa was allowed to react with a substrate and a test compound in a 30 mmol/L HEPES buffer solution (pH 7.4) that contained 145 mmol/L NaCl, 5 mmol/L KCl, and 1 mg/mL PEG8000 (manufactured by QIAGEN) at 37° C. for 10 minutes. To the substrate, S-2366 (pyroGlu-Pro-Arg-pNA.HCl) was added to a final concentration of 300 μmol/L. After the reaction had progressed, the absorbance at OD 405 nm was measured. A change in OD without addition of the test compound was set at 100%, and a concentration at which an increase in the OD value is suppressed by 50% was calculated as an IC50 value (nmol/L).
Measurement: IC50 = 285 nM (pChEMBL 6.539999961853027)
Descriptors: MW 592.11, ALogP 5.25, HBA 6, HBD 3, PSA 135.1, QED 0.38
Compound (SMILES): C[C@H](NC(=O)c1cc(C#N)cnc1NCc1ccc(-c2cnc(N)c(C(=O)NCCN(C)C)n2)cc1)c1ccc(F)cc1
InChIKey: GYCRMLBPKGXHNU-IBGZPJMESA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: PDK1 kinase assay: PDK1 (amino acids 51-359) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to homogeneity. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 4-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide (final concentrations: 0.75 nM PDK1, 10 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 40 μM of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm. The fluorescence polarization value P is defined by the equation below. The value mP is generated by multiplying the P value for each reaction well by a factor of 1000. The value ΔmP for each well is the mP value for that well minus the mP value for the average negative control. P=(Fpar−Fperp)/(Fpar+Fperp)  Eq.:Where par is fluorescence intensity parallel to the excitation plane; and perp is fluorescence intensity perpendicular to the excitation plane. ΔmP values were plotted as a function of compound concentration and the data were analyzed with a 4-parameter fit using GraphPad Prism software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 581.66, ALogP 3.53, HBA 9, HBD 4, PSA 161.95, QED 0.21
Compound (SMILES): Nc1ncnc(N2CC[C@H](O)[C@@H](N3CCCC(Nc4cc(F)cc(Cl)c4)C3=O)C2)c1F
InChIKey: RSCJXGAPQJLFES-YVZMLIKISA-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 < 100 nM (pChEMBL 7)
Descriptors: MW 452.89, ALogP 2.03, HBA 7, HBD 3, PSA 107.61, QED 0.65
Compound (SMILES): CC(=O)N(C)C[C@@H](Nc1ncc(-c2nnc(C(F)F)o2)cn1)c1ccc(F)cc1
InChIKey: OXHAMRNZBAQLSJ-CQSZACIVSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Enzymatic Activity Assay: All enzymatic reactions were conducted in duplicate at room temperature for 17 hours in a 50 μl mixture containing HDAC assay buffer (50 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, 5 μg BSA), an HDAC substrate, an HDAC enzyme, and a test compound. Compound dilution was prepared ten-fold higher than the final concentration of the compounds with 10% DMSO in HDAC assay buffer and 5 μl of the dilution was added to a 50 μl reaction so that the final concentration of DMSO is 1% in all of reactions. After enzymatic reactions, 50 μl of HDAC Developer was added to each well and the plate was incubated at room temperature for an additional 20 minutes. Fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm using a Tecan Infinite M1000 or Biotek Synergy microplate reader.The fluorescent intensity data were analyzed using the computer software, Graphpad Prism. In the absence of the compound, the fluorescent intensity (Ft) in each data set was defined as 100% activity. In the absence of HDAC, the fluorescent intensity (Fb) in each data set was defined as 0% activity. The percent activity in the presence of each compound was calculated according to the following equation: % activity=(F−Fb)/(Ft−Fb), where F=the fluorescent intensity in the presence of the compound.
Measurement: IC50 = 360 nM (pChEMBL 6.440000057220459)
Descriptors: MW 406.37, ALogP 3.23, HBA 7, HBD 1, PSA 97.04, QED 0.64
Compound (SMILES): CNC1OC(c2ccc(Cl)c(Cl)c2)=NC1C#N
InChIKey: JXRIWCZBEQSBPK-UHFFFAOYSA-N
Target: Polyunsaturated fatty acid lipoxygenase ALOX15 (SINGLE PROTEIN), Homo sapiens
UniProt: P16050
Assay: Lipoxygenase UV-Vis Assay: The inhibitor compounds were screened initially using one concentration point on a Perkin-Elmer Lambda 40 UV-Vis spectrometer. The percent inhibition was determined by comparing the enzyme rates of the control (DMSO solvent) and the inhibitor sample by following the formation of the conjugated diene product at 234 nm (ε=25,000 M−1 cm−1). The reactions were initiated by adding either of 40 nM 12-LOX, 40 nM 12/15-LOX, 0.5 μM 15-LOX-2 or 200 nM 5-LOX (ammonium sulfate suspension) to a cuvette with a 2 mL reaction buffer constantly stirred using a magnetic stir bar at room temperature (22° C.). It should be noted that LOX isozymes are often expressed in the inactive demetallated form, so it is best to utilize activity to determine the optimal LOX concentration for the assay (optimal rate of approximately 0.001 abs/sec at 10 μM AA). Reaction buffers used for various lipoxygenase were as follows-25 mM HEPES (pH 7.3), 0.3 mM CaCl2, 0.1 mM EDTA, 0.2 mM ATP, 0.01% Triton X-100, 10 μM AA for the crude, ammonium sulfate precipitated 5-LOX; 25 mM Hepes (pH 8), 0.01% Triton X-100, 10 μM AA for 12-LOX and 25 mM Hepes buffer (pH 7.5), 0.01% Triton X-100, 10 μM AA for 12/15-LOX and 15-LOX-2. The substrate concentration was quantitatively determined by allowing the enzymatic reaction to go to completion in the presence of 15-LOX-2. For the inhibitors that showed more than 50% inhibition at the one point screens (25 μM inhibitor), IC50 values were obtained by determining the enzymatic rate at various inhibitor concentrations and plotted against inhibitor concentration (Approximate range: 0.1 to 25 μM inhibitor), followed by a hyperbolic saturation curve fit (assuming total enzyme concentration [E]<<Ki app, so IC50Ki app). It should be noted that all of the potent inhibitors displayed greater than 80% maximal inhibition, unless stated in the tables. Inhibitors were stored at −20° C. in DMSO.
Measurement: IC50 = 810 nM (pChEMBL 6.090000152587891)
Descriptors: MW 270.12, ALogP 2.21, HBA 4, HBD 1, PSA 57.41, QED 0.9
Compound (SMILES): CC(C)c1cc(C(=O)N2CCC(NC(=O)C3CCOC3)C2)[nH]n1
InChIKey: IKHRJJHJJBKHLU-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5A (SINGLE PROTEIN), Homo sapiens
UniProt: P29375
Assay: KDM5 Enzyme Assay: Full length KDM5A enzyme was expressed and purified inhouse. Biotin-H3K4me3 peptide was purchased from New England Biolabs. HTRF reagents (containing Eu-labeled H3K4me1-2 antibody, and streptavidin-XL665) were purchased from Cis-Bio International. Plates were read on an Envision multi-label plate reader (Perkin Elmer).The HTRF assay mixture contained 2 nM full length KDM5A enzyme, 100 nM H3K4Me3 peptide substrate, 1 uM 2-OG, 100 uM Fe2+, 500 uM ascorbate. 50 mM HEPES pH 7.0 buffer, 0.01% Triton-X 100, 2 mM DTT, 0.25% DMSO at a final volume of 10 uL.The enzyme reaction was carried out at room temperature in black Proxiplate 384-Plus plate (Corning. Costar) within 30 minutes, in the presence of varying concentration of a test compound. At the end of enzyme reaction, 5 uL of 1 mM EDTA were added to quench the reaction and then the detection reagents (5 uL) were added to give final concentrations of 0.5 nM Eu-labeled H3K4me1-2 antibody, and 50 nM streptavidin-XL665. The plates were incubated at room temperature for 60 minutes and then read in the Envision plate reader. The readouts were transformed into % inhibition, and IC50 value of a test compounds was generated by using four parameters curve fitting (Model 205 in XLFIT5, iDBS).
Measurement: IC50 = 0.12 nM (pChEMBL 9.920000076293945)
Descriptors: MW 320.39, ALogP 0.9, HBA 4, HBD 2, PSA 87.32, QED 0.86
Compound (SMILES): Cn1nnnc1Cn1cnc2c(N3CCC(F)(F)C3)nc(C(C)(C)C)nc21
InChIKey: PYAGZJNEPGFVJA-UHFFFAOYSA-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), 1x 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 ul 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 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM 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 uM 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 uM 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 uM and selectivity versus CB1 in the corresponding assay of at least 500 fold.
Measurement: EC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 377.4, ALogP 1.54, HBA 9, HBD 0, PSA 90.44, QED 0.68
Compound (SMILES): COc1nn(C)cc1Nc1ncc(F)c(-c2c[nH]c3c(NC(=O)[C@@H](C)N4CCN(C)[C@H](C)C4)cccc23)n1
InChIKey: HKMXSVZYKLTDAP-HZPDHXFCSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzymatic Assay: Enzyme inhibition studies were performed using recombinant JAK1 (amino acids 866-1154, Life Technologies, #PV4774, Carlsbad, Calif.), JAK2 (amino acids 831-1132), or JAK3 (amino acids 781-1124) under buffer conditions of 50 mM HEPES pH 7.3, 1 mM DTT, 0.01% Tween® 20, 50 μg/mL BSA, and 10 mM MgCl2. JAK enzyme was expressed as an N-terminal GST fusion in insect cells and purified by glutathione-affinity and size-exclusion chromatographies. Enzymes were assayed both at their respective ATP Km (JAK1: 55 μM, JAK2: 15 μM, JAK3: 3 μM) and the approximated high end of physiological ATP concentration of 5 mM, in the presence of inhibitor dosed at 30, 3, 0.3, 0.03, 0.003 and 0 μM final test concentrations. For JAK1, 6 nM of enzyme (for Km ATP assay) or 4 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (FITC-C6-KKHTDDGYMPMSPGVA-NH2 (SEQ ID NO:1), Intonation, Boston, Mass.). For JAK2, 0.8 nM of enzyme (for Km ATP assay) or 0.3 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). For JAK3, 0.2 nM of enzyme (for Km ATP assay) or 0.1 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). Phosphorylated and unphosphorylated peptides were separated and quantified by a Caliper LC3000 system (Caliper Life Sciences, MA) for calculating percent inhibition. The results of this assay are shown in Table 16 and indicate that the compounds of Formula (I), (Ia), (Ib) and Table 1 exhibit preferential inhibition of JAK1 over JAK2 (in many cases demonstrating over 100 times selectivity for inhibition of JAK1 vs. JAK2).
Measurement: IC50 = 85 nM (pChEMBL 7.070000171661377)
Descriptors: MW 521.6, ALogP 3.21, HBA 9, HBD 3, PSA 116.23, QED 0.34
Compound (SMILES): Cc1cc(C#N)ccc1-c1cnn(-c2ccc(C(=O)N3CCN(C)CC(F)(F)C3)cn2)c1O
InChIKey: YBAHSUMYFQBXPY-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Inhibition Assay: The IC50 values for the PHD2 enzyme (residues 181-417) were determined by mixing increasing amounts of inhibitor with a fixed amount of enzyme (5 nM, final concentration) and Biotin labeled peptide (Biotin-Asp-Leu-Glu-Met-Leu-Ala-Pro-Tyr-Ile-Pro-Met-Asp-Asp-Asp-Phe-Gln-Leu, 1 uM final concentration) and 2-Oxyglutarate (2 uM final concentration) in 50 mM HEPES, 50 mM KCl, 0.5 mM TCEP, 2 uM FeCl2, 0.1 mg/ml BSA, at pH 7.3. The reaction was conducted by pre-incubating the enzyme in the presence of inhibitor for 60 min at room temperature. The activity of the free enzyme was measured by adding the peptide, the 2-Oxoglutarate (see above for final concentrations), and Ascorbic Acid (1 mM final concentration). The enzymatic activity was quenched after 60 min by adding an excess of a tight binding inhibitor to the assay mixture. The amount of product released was measured by using a LC/MS system (Agilent HPLC with Applied Biosystems API3000 Mass Spectrometer).
Measurement: IC50 = 3.47 nM (pChEMBL 8.460000038146973)
Descriptors: MW 452.47, ALogP 2.84, HBA 7, HBD 1, PSA 98.28, QED 0.66
Compound (SMILES): COc1ncc(-c2nc(N3CCOCC3)nc3c(CN4CC5CC5C4)c(C)sc23)cc1NS(C)(=O)=O
InChIKey: OEHJVHQPQLXNAO-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Enzyme Activity Inhibition Assay: 1. Buffer preparation: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS.2. Compound was formulated in 100% DMSO in a concentration gradient, and added to a 384-well plate to a final DMSO concentration of 1%.3. PI3Kα, PI3Kδ, PI3Kβ and PI3Kγ enzymes were diluted to an optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT, and transferred to a 384-well plate and incubated with the compound for a certain time.4. Substrate was diluted to an optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT, 50 μM PIP2, 25 μM ATP; then added to a 384-well plate to initiate the reaction, and for PI3Kα, PI3Kβ and PI3Kγ at room temperature reacted for 1 hr, for PI3Kδ at room temperature reacted for 2 hrs. For PI3Kβ and PI3Kγ, it was still necessary to further add 10 μL ADP-Glo Detection Reagent, and then equilibrated at room temperature for 30 minutes.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 530.68, ALogP 2.73, HBA 10, HBD 1, PSA 109.78, QED 0.49
Compound (SMILES): CCNc1nc(Nc2cn(S(C)(=O)=O)nc2C2CC2)ncc1Br
InChIKey: UZFXAPHBFKMVKN-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Biochemical Assay: Materials:    LRRK2 G2019S enzyme    Substrate (LRRKtide)    ATP    TR-FRET dilution buffer    pLRRKtide antibody    384-well assay plate    DMSOEnzyme Reaction Conditions    50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, 2 mM DTT    5 nM LRRK2    134 μM ATP    60 minute reaction time    23° C. reaction temperature    10 μL total reaction volumeDetection Reaction Conditions    1× TR-FRET dilution buffer    10 mM EDTA    2 nM antibody    23° C. reaction temperature    10 μL total reaction volumeCompounds were prepared by initially diluting to 1 mM with DMSO. 35 μL of reference compound solution, 35 μL of test compound solution, and 35 μL HPE were successively added to the source plate (384-well assay plate, Labcyte). The plates were centrifuged at 2500 rpm for 1 minute and sealed in foil. POD was used to perform a 3.162 fold serial dilution and 100 nL of reference compound solution, test compound solution, HPE and ZPE were transferred to assay plates. The assay plate was centrifuged at 2500 rpm for 1 minute, and sealed with foil.To perform the enzyme reaction, 5 μL of LRRKtide substrate and kinase mixture in assay buffer was added to all wells of the assay plate. The plate was centrifuged to concentrate the mixture at the bottom of the wells. The assay plate was incubated at 23° C. for 20 minutes. Following incubation, 5 μL of 2× ATP in assay buffer was added to each well, and plates were centrifuged to concentrate the mixture at the bottom of the wells. The plate was incubated at 23° C. for 60 minutes.To perform the detection of the reaction, EDTA completely mixed in TR-FRET dilution buffer was added to antibody reagent. 10 μL of detection reagent was added to all wells of each well of the assay plate and the plate was centrifuged to concentrate the mixture at the bottom of the wells. The plate was then incubated at 23° C. for 60 minutes. Plates were read on Perkin Elmer Envision 2104 instrument in TR-FRET mode using a 340 nm excitation filter, 520 nm fluorescence emission filter, and 490 or 495 nm terbium emission filter.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 401.29, ALogP 2.3, HBA 8, HBD 2, PSA 101.8, QED 0.77
Compound (SMILES): O=S(=O)(c1ccc(F)cc1)C1(c2ccc(C(Oc3ccc(F)cc3)(C(F)(F)F)C(F)(F)F)cc2)CCCC1
InChIKey: UNRSJOVZOZFVNJ-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: 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) binding assay. 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 chromotography. 15 ug/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 room temperature 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).
Measurement: IC50 = 1298 nM (pChEMBL 5.889999866485596)
Descriptors: MW 564.49, ALogP 7.61, HBA 3, HBD 0, PSA 43.37, QED 0.23
Compound (SMILES): Nc1ncnn2c(-c3cccc(N4CCN(c5ncccn5)CC4=O)c3)cc(-c3ccnn3C3CCOCC3)c12
InChIKey: HUVYDQLFNFXCOP-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assay: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. The IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 536.6, ALogP 2.84, HBA 11, HBD 1, PSA 132.59, QED 0.36
Compound (SMILES): CCC(O)c1cc(Nc2nc(N3CCC[C@@H](N)C3)ncc2C(N)=O)cc(C(C)(C)C)c1
InChIKey: JTWDPICHSFLTJA-VTBWFHPJSA-N
Target: Calcium/calmodulin-dependent protein kinase type 1D (SINGLE PROTEIN), Homo sapiens
UniProt: Q8IU85
Assay: Biochemical CaMK1D Enzymatic Activity Assay: The following describes an ADP-Glo Kinase Assay, which measures the ADP formed from a kinase reaction; the ADP generated is converted into ATP and is used to generate light in a luciferase reaction. The assay is used to assess the effect of compounds on the activity of purified CaMK1 D.Materials and Solutions:All reagents are from Sigma-Aldrich unless otherwise specified. ADP-Glo Kinase Assay (Promega, V9102). His-tagged CaMK1D_1-385 (Fisher Scientific, PR6770A). Autocamtide-2 (SignalChem, A15-58). Calmodulin (Merck, 208694). 1M Tris-HCl pH7.5 (Fisher Scientific, 10123722). 1M DTT (Fisher Scientific, 10674545). Calcium chloride (C1016). Magnesium Chloride (M8266). DMSO (D8418). Autocamtide-2 provided as a lyophilised powder and prepared as a 10 mM stock in MilliQ water. RB: 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1 CaCl2), just prior to use 1M DTT was added to a final concentration of 2 mM.Assay Protocol: 7.88 μL reaction mixture (including: calmodulin, Autocamtide-2, CaMK1 D in RB) was incubated with 12 μL test compound in 100% DMSO. To start the reaction 4 μL of ATP mixture were added. Final assay concentrations: 3 nM CaMK1D, 1 μM Calmodulin, 125 μM Autocamtide-2 and 10 μM ATP. Plates were incubated at 25C for 2 hours prior to the 1:1 addition of ADP-Glo reagent. Plates were incubated for a further 1 hour prior to the 1:1 addition of ADP-Glo substrate. After 30 minutes plates read with the EnVision® Multilabel Plate Reader, using Luminescence 700. Compound IC50 was determined using a 4-parameter equation and are reported in nM.
Measurement: IC50 = 2287 nM (pChEMBL 5.639999866485596)
Descriptors: MW 426.57, ALogP 2.99, HBA 7, HBD 4, PSA 130.39, QED 0.56
Compound (SMILES): COc1ccc(C2COc3c(C)c(Cn4cnc5cc(C#CC(C)(C)N)cnc54)c(C)c(F)c3O2)cn1
InChIKey: SRUZFHAIPXHYLO-UHFFFAOYSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07333
Assay: In Vitro Studies: All assay reaction conditions for IC50 determinations were within the linear range with respect to time and enzyme concentration. In a 384 well polypropylene plate, c-FMS (0.14 nM, Carna 08-155) was pre-incubated in a 100 mM Hepes-NaOH pH 7.5 buffer containing 0.01% Triton X-100, 10 mM MgCl2, 0.1% BSA, 1 mM DTT, 10 μM sodium orthovanadate and 10 μM beta-glycerophosphate and compound with a concentration of 2.5% DMSO for 15 minutes at room temperature. The reaction was initiated with an equal volume of peptide substrate (Caliper Life Sciences catalog no. 760430) and ATP in the aforementioned buffer. The final concentrations in the reaction were 70 pM c-FMS, 1.5 μM peptide substrate and 500 μM ATP (ATP Km). The reaction was incubated at room temperature for 120 minutes and terminated with a buffer containing excess EDTA (100 mM Hepes-NaOH pH 7.5, 0.02% Brij, 0.1% CR-3, 0.36% DMSO and 100 mM EDTA). The plate was run for one cycle on a LabChip 3000 (Caliper Life Sciences, Hopkinton, Mass.) in an off-chip mobility shift type assay with an upstream voltage of −2250 volts, a downstream voltage of −500 volts and a vacuum pressure of −1.6 psi.
Measurement: IC50 = 383 nM (pChEMBL 6.420000076293945)
Descriptors: MW 501.56, ALogP 4.24, HBA 8, HBD 1, PSA 97.31, QED 0.42
Compound (SMILES): O=C(NCCCN1CCOCC1)c1cnccc1Nc1nc(-c2cccc(C(F)(F)F)n2)nn2cccc12
InChIKey: RTMOMQLZSDSJRU-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assays: Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of 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 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 526.52, ALogP 3.4, HBA 9, HBD 2, PSA 109.57, QED 0.34
Compound (SMILES): CC1(C)[C@H]2CC[C@]1(c1ccnc(-n3cnc(C[C@H](O)CO)n3)n1)c1nnc(-c3c(F)cccc3F)cc12
InChIKey: OISULXUVZANVJE-GSLFCAENSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Ligand Binding Assay: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2; 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 uM 25-hydroxycholesterol was also present. Assay plates were 96-well polypropylene V-bottom plates. 10 uL of 5× compound in 25% DMSO/75% Assay Buffer was added to Test wells. 10 uL of 25% DMSO/75% Assay Buffer was added to Total Binding or No Receptor wells. 10 uL of 5 uM 25-hydroxycholesterol in 25% DMSO/75% Assay Buffer was added to NSB wells. 20 uL of 15 nM 25-[3H]hydroxycholesterol prepared in Assay Buffer was added to all wells. 20 uL of 1.5 ug/mL RORc receptor was added to wells (or 40 uL Assay Buffer to No R wells). Following addition to the wells, the plates were incubated 3 h at 25° C. Filtration
Measurement: IC50 = 53 nM (pChEMBL 7.28000020980835)
Descriptors: MW 505.53, ALogP 2.89, HBA 9, HBD 2, PSA 122.73, QED 0.41
Compound (SMILES): CNC1OC(c2c[nH]c3ccccc23)=NC1C#N
InChIKey: VXCSAQHRDHNBEX-UHFFFAOYSA-N
Target: Polyunsaturated fatty acid lipoxygenase ALOX15 (SINGLE PROTEIN), Homo sapiens
UniProt: P16050
Assay: Lipoxygenase UV-Vis Assay: The inhibitor compounds were screened initially using one concentration point on a Perkin-Elmer Lambda 40 UV-Vis spectrometer. The percent inhibition was determined by comparing the enzyme rates of the control (DMSO solvent) and the inhibitor sample by following the formation of the conjugated diene product at 234 nm (ε=25,000 M−1 cm−1). The reactions were initiated by adding either of 40 nM 12-LOX, 40 nM 12/15-LOX, 0.5 μM 15-LOX-2 or 200 nM 5-LOX (ammonium sulfate suspension) to a cuvette with a 2 mL reaction buffer constantly stirred using a magnetic stir bar at room temperature (22° C.). It should be noted that LOX isozymes are often expressed in the inactive demetallated form, so it is best to utilize activity to determine the optimal LOX concentration for the assay (optimal rate of approximately 0.001 abs/sec at 10 μM AA). Reaction buffers used for various lipoxygenase were as follows-25 mM HEPES (pH 7.3), 0.3 mM CaCl2, 0.1 mM EDTA, 0.2 mM ATP, 0.01% Triton X-100, 10 μM AA for the crude, ammonium sulfate precipitated 5-LOX; 25 mM Hepes (pH 8), 0.01% Triton X-100, 10 μM AA for 12-LOX and 25 mM Hepes buffer (pH 7.5), 0.01% Triton X-100, 10 μM AA for 12/15-LOX and 15-LOX-2. The substrate concentration was quantitatively determined by allowing the enzymatic reaction to go to completion in the presence of 15-LOX-2. For the inhibitors that showed more than 50% inhibition at the one point screens (25 μM inhibitor), IC50 values were obtained by determining the enzymatic rate at various inhibitor concentrations and plotted against inhibitor concentration (Approximate range: 0.1 to 25 μM inhibitor), followed by a hyperbolic saturation curve fit (assuming total enzyme concentration [E]<<Ki app, so IC50Ki app). It should be noted that all of the potent inhibitors displayed greater than 80% maximal inhibition, unless stated in the tables. Inhibitors were stored at −20° C. in DMSO.
Measurement: IC50 = 3900 nM (pChEMBL 5.409999847412109)
Descriptors: MW 240.27, ALogP 1.38, HBA 4, HBD 2, PSA 73.2, QED 0.83
Compound (SMILES): c1ccc([C@H]2C[C@@H]2NC2CNC2)cc1
InChIKey: ALKULJHQEDUCEU-NEPJUHHUSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: Biological Assay: Human recombinant monoamine oxidase proteins MAO-A and MAO-B were purchased from Sigma Aldrich. MAOs catalyze the oxidative deamination of primary, secondary and tertiary amines. In order to monitor MAO enzymatic activities and/or their inhibition rate by inhibitor(s) of interest, a fluorescence-based (inhibitor)-screening assay was set up. 3-(2-Aminophenyl)-3-oxopropanamine (kynuramine dihydrobromide, Sigma Aldrich), a non fluorescent compound was chosen as a substrate. Kynuramine is a non-specific substrate for both MAO-A and MAO-B activities. While undergoing oxidative deamination by MAO activities, kynuramine is converted into 4-hydroxyquinoline (4-HQ), a resulting fluorescent product.The monoamine oxidase activity was estimated by measuring the conversion of kynuramine into 4-hydroxyquinoline. Assays were conducted in 96-well black plates with clear bottom (Corning) in a final volume of 100 uL. The assay buffer was 100 mM HEPES, pH 7.5. Each experiment was performed in duplicate within the same experiment. Briefly, a fixed amount of MAO (0.25 ug for MAO-A and 0.5 ug for MAO-B) was incubated on ice for 15 minutes in the reaction buffer, in the absence and/or in the presence of at least eight 3-fold serial dilutions each. Clorgyline and Deprenyl (Sigma Aldrich) was used as a control for specific inhibition of MAO-A and MAO-B respectively. After leaving the enzyme(s) interacting with the inhibitor, KM of kynuramine was added to each reaction for MAO-B and MAO-A assay respectively, and the reaction was left for 1 hour at 37° C. In the dark. The oxidative deamination of the substrate was stopped by adding 50 uL of NaOH 2N. The conversion of kynuramine to 4-hydroxyquinoline, was monitored by fluorescence (excitation at 320 nm, emission at 360 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure levels of fluorescence produced in the absence and/or in the presence of inhibitor. The maximum of oxidative deamination activity was obtained by measuring the amount of 4-hydroxyquinoline formed from kynuramine deamination in the absence of inhibitor and corrected for background fluorescence in the absence of MAO enzymes. The IC50 values of each inhibitor were calculated with GraphPad Prism Software.
Measurement: IC50 = 23750 nM (pChEMBL 4.619999885559082)
Descriptors: MW 188.27, ALogP 1.1, HBA 2, HBD 2, PSA 24.06, QED 0.74
Compound (SMILES): CC1=C(C)Cc2cc(C(=O)NCc3c(C)cc(C)[nH]c3=O)cc(Br)c2OC1
InChIKey: JKOXAERUTDGGMY-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Biological Assay: EZH2 Histone Methyl Transferase Assay: The effectiveness of compounds of the present invention as inhibitors of histone methyl transferases can be readily tested by assays known to those skilled in the art. For example, in vitro histone methyl transferase assays may be conducted with a relevant purified histone methyl transferase and an appropriate synthetic substrate to determine the inhibitory activity of the compounds. Assays for inhibition of EZH2 by the instant compounds were performed in 384-well plates with reaction mixtures containing 350 nM of histone peptide substrate (ATKAAR-K(Me2)-SAPATGGVKKPHRYRPG-GK(Biotin), 500 nM S-[methyl-3H]adenosyl-L-methionine (55-85 Ci/mmol), 50 mM Tris-HCl (pH 9.0), 50 mM NaCl, 1 mM dithiothreitol, Tween-20 at 0.01% and fatty-acid free bovine serum albumin at 0.01%, and recombinant EZH2-641F complex (EZH2 Y641F/EED/SUZ12/RbAp48/AEBP2) at 5 nM (>98% purity, BPS Bioscience) or 15 nM (50% purity, in-house). Reaction mixtures were incubated at room temperature for 3 hours, and the reactions were terminated by 0.005% poly-L-lysine solution in 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl. Reaction products were captured by binding to strepavidin-conjugated imaging beads. Incorporation of radioactive methyl group into the histone peptide substrate was determined in Leadseeker (GE Healthcare) by means of scintillation proximity assay.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 431.33, ALogP 3.96, HBA 3, HBD 2, PSA 71.19, QED 0.72
Compound (SMILES): CC1(C)OCCn2c1nc1c(C3CC3)nc(-c3cnc(N)c(C#N)c3)nc12
InChIKey: LZIDZKAPYXYGJI-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 12 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12852
Assay: DLK TR-FRET Inhibition Assay: DLK kinase reactions (20 μL) containing 5 nM N-terminally GST-tagged DLK (catalytic domain amino acid 1-520) (Carna Bioscience), 40 nM N-terminally HIS tagged MKK4 K131M substrate, and 30 μM ATP in kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Triton X-100, 0.01% Bovine γ-Globulins, 2 mM DTT, 10 mM MgCl2 and 1 mM EGTA), and testing compound 1:3 serial diluted starting at 20 uM were incubated at ambient temperature for 60 minutes in 384 well OptiPlate (Perkin Elmer). To quench kinase reactions and detect phosphorylated MKK4, 15 μL of TR-FRET antibody mixture containing 2 nM anti-phosphorylated MKK4 labeled with Europium cryptate (Cisbio) and 23 nM anti-HIS labeled with D2 (Cisbio) in detection buffer (25 mM Tris pH 7.5, 100 mM NaCl, 100 mM EDTA, 0.01% Tween-20, and 200 mM KF) was added to the reaction mixture. The detection mixture was incubated for 3 hours at ambient temperature and the TR-FRET was detected with an EnVision multilabel plate reader (Perkin-Elmer) using the LANCE/DELFIA Dual Enh label from Perkin-Elmer (excitation filter: UV2 (TRF) 320 and emission filters: APC 665 and Europium 615).
Measurement: Ki = 264 nM (pChEMBL 6.579999923706055)
Descriptors: MW 361.41, ALogP 2.48, HBA 8, HBD 1, PSA 115.53, QED 0.75
Compound (SMILES): OC(CC1c2ccccc2-c2cncn21)C1CCC(C(F)(F)F)CC1
InChIKey: DXHZTNYHUDMHMF-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzymatic IDO Assay: The IC50 values for each compound were determined by testing the activity of IDO in a mixture containing 50 mM potassium phosphate buffer at pH 6.5; 70 nM purified human IDO protein, 200 μM L-tryptophan, 20 mM ascorbate, 20 μM methylene blue, 0.1% DMSO. The inhibitors were initially diluted in DMSO at 100 mM and were diluted in potassium phosphate 50 mM, added to the reaction mixture at final concentrations raging from 1 mM to 5 nM and preincubated with the enzyme for 5 min at 25° C. The reaction was started by addition of L-tryptophan to 200 μM and incubated 15 min at 37° C. The reaction was stopped by addition of 0.5 vol of 30% trichloroacetic acid and incubated 30 min at 60° C. to hydrolyze N-formylkynurenine to kynurenine. The reaction was centrifuged at 3400 g for 5 min to remove precipitated protein and the supernatant was reacted with 2% (w/v) of p-dimethylaminobenzaldehyde in acetic acid. The reaction was incubated 10 min at 25° C. and read at 480 nm in a spectrophotometer. Control samples with no IDO inhibitor, or with no IDO enzyme or with the reference inhibitors 1-methyl-tryptophan (200 μM) and menadione (1.2 μM) were used as controls to set the parameters for the non-linear regressions necessary for determination of the IC50 for each compound. Nonlinear regressions and determination of the IC50 values were performed using the GraphPad Prism 4 software. Compounds with an IC50 of less than 500 μM were considered as active inhibitors in this assay.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 350.38, ALogP 4.57, HBA 3, HBD 1, PSA 38.05, QED 0.88
Compound (SMILES): CCC(=O)N[C@H]1CC[C@@H](N(C)C(=O)c2ccc(-c3ccc(Cl)cc3F)cc2)C1
InChIKey: CTSMRVKEHWTUCA-ZWKOTPCHSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay of FASN: The biochemical FASN activity testing was performed as 384 well two-time-point kinetic fluorescence intensity assay format in Greiner low volume medium binding 384-well black microtiter plates in a total assay volume of 8 μl and was used for high throughput screen. In each well 3 μl 40 nM human recombinant full-length fatty acid synthase (produced in-house in SF9 cells) were dispensed in the following assay buffer: 50 mM potassium phosphate buffer pH 7.0, 0.005% (w/v) BSA, 2 mM Glutathione, 0.02% Tween-20. Then 2 μl of 200 μM NADPH in assay buffer were added, followed by the addition of the test compounds in 10 dilution concentrations starting with 30 μM (final concentration) to get a final DMSO content of 1% (v/v). The mixture was incubated for at least 15 min at room temperature. After the pre-incubation the enzymatic reaction was started by the addition of 2 μl substrate solution (80 μM acetyl-CoA, 240 μM malonyl-CoA). A first fluorescence intensity measurement (time point one) was performed with an Envision multimode reader (Perkin Elmer LAS Germany GmbH) at excitation wavelength 340 nm (lamp mode) and emission wavelength 460 nm. The reaction was incubated for 30 minutes at room temperature. After this the fluorescence intensity was measured again in the Envision using the same parameters as described above (second time point measurement). The data were analysed by subtracting the first time point measurement value from the second time point measurement value (after the enzymatic reaction). The differences of the emission signals were determined. These reflect directly the conversion rate of NADPH. The full value used was the inhibitor-free reaction. A pharmacological zero value was used like GSK837149A (Sigma-Aldrich) in a final concentration of 5-10 μM. The inhibitory values (IC50) were determined using either the program Symyx Assay Explorer® or Condosseo® from GeneData.
Measurement: IC50 < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 402.9, ALogP 4.67, HBA 2, HBD 1, PSA 49.41, QED 0.79
Compound (SMILES): CCCOc1nc(N2CCC(F)(F)C2)c2nnn(Cc3ccccc3Cl)c2n1
InChIKey: TUUMVNRPSMSYFO-UHFFFAOYSA-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.
Measurement: EC50 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 408.84, ALogP 3.56, HBA 7, HBD 0, PSA 68.96, QED 0.62