lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cncc(-c2cc3c(cn2)cnn3-c2cccc(N3CC[C@H](N)C3)n2)n1
InChIKey: WJXBLZWJWGLQLR-HNNXBMFYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.34 nM (pChEMBL 9.470000267028809)
Descriptors: MW 372.44, ALogP 2.12, HBA 8, HBD 1, PSA 98.64, QED 0.59
Compound (SMILES): COc1cn(C(CC2(C)COC2)C(=O)Nc2ccc(C(=O)O)cc2)c(=O)cc1-c1cc(Cl)ccc1C#N
InChIKey: UCSCLOQQIVMMIJ-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Measurement of FXIa Inhibition: To determine the factor XIa inhibition of the substances according to the invention, a biochemical test system is used which utilizes the reaction of a peptidic factor XIa substrate to determine the enzymatic activity of human factor XIa. Here, factor XIa cleaves from the peptic factor XIa substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm). The measured emissions of the test batches with test substance are compared to those of control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide), and the IC50 values are calculated from the concentration/activity relationships.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 521.96, ALogP 4.35, HBA 7, HBD 2, PSA 130.65, QED 0.45
Compound (SMILES): C[C@@H]1CN(Cc2ccccc2)c2ncccc2[C@H](C)N1C(=O)NCc1ccccc1
InChIKey: FZSRPXVMKCEYMK-UXHICEINSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1I (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P0X4
Assay: Channel blocking activity at recombinant human CaV 3.3 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 400.53, ALogP 4.76, HBA 3, HBD 1, PSA 48.47, QED 0.69
Compound (SMILES): Cc1cc(NC(=O)N2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)sn1
InChIKey: KSIKQWKOYHAWGR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 382.45, ALogP 2.78, HBA 7, HBD 2, PSA 104.1, QED 0.55
Compound (SMILES): NC1CN(c2cccc(-n3ncc4cnc(-c5cnn(CC(F)(F)F)c5)cc43)n2)C1
InChIKey: OVTVOGQQMFPUDP-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 1.14 nM (pChEMBL 8.9399995803833)
Descriptors: MW 414.4, ALogP 2.39, HBA 8, HBD 1, PSA 90.68, QED 0.55
Compound (SMILES): O=C(c1cc(F)ccc1-n1nccn1)N1CCOC[C@H]1Cc1cccc(-n2cccn2)c1
InChIKey: SYLDJPVOGVSYAI-HXUWFJFHSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: CYP3A4 Inhibition Assay: The CYP3A4 inhibition assay was performed using human liver microsomes and testosterone 6'-hydroxylation as a P450 isoform-specific marker. In a total volume of 150 uL, 14C-testosterone at a final concentration of 40 uM in a 100 mM phosphate buffer (pH 7.4) was incubated in a 96-well plate with 0.3 mg/mL of human liver microsomes in an Eppendorf thermomixer at 37° C. and 400 rpm. A 1.0 uL-aliquot of the 150-fold concentrated compound stock solution, prepared in DMSO, was added to yield final inhibitor concentrations of 0, 0.1, 0.5, 1.0, 5.0, 10, 25, and 50 uM. The reaction was initiated by addition of 15 uL of the NADPH-regenerating system containing the glucose-6-phosphate dehydrogenase and terminated after 7 min with a 75 uL-aliquot of methanol. After centrifugation at 465 g and 4° C. for 20 min, a 50 uL-aliquot of the supernatant was submitted to HPLC according to the method described below.
Measurement: IC50 = 25000 nM (pChEMBL 4.599999904632568)
Descriptors: MW 432.46, ALogP 2.68, HBA 7, HBD 0, PSA 78.07, QED 0.48
Compound (SMILES): O=C(O)COC[C@H]1CC[C@H](COC(=O)N(c2cccc(F)c2)c2ccc(Cl)c(F)c2)CC1
InChIKey: IONAKCRDZACAPF-WKILWMFISA-N
Target: Prostacyclin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43119
Assay: Agonist activity at recombinant human IP receptor expressed in CHO-K1 cells assessed as increase in intracellular cAMP level after 1 hr incubation by HTRF method
Measurement: EC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 467.9, ALogP 5.8, HBA 4, HBD 1, PSA 76.07, QED 0.53
Compound (SMILES): [C-]#[N+]c1c(F)ccc(C2CN3CCN(C(=O)Cc4ccc(-n5cnnn5)nc4)CC3CCO2)c1C
InChIKey: YYLKTLHBSVNPLY-UHFFFAOYSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Thallium Flux Assay: The ROMK channel functional thallium flux assay is performed in 384 wells, using the FLIPR-Tetra instrument. HEK-hKir1.1 cells are seeded in Poly-D-Lysine microplates and kept in a 37° C.-10% CO2 incubator overnight. On the day of the experiment, the growth media is replaced with the FluxOR™ reagent loading buffer and incubated, protected from light, at ambient temperature (23-25° C.) for 90 min. The loading buffer is replaced with assay buffer±test compound followed by 30 min incubation at ambient temperature, where the Thallium/Potassium stimulant is added to the microplate. The fluorescence intensity of wells containing 3 μM of a standard control ROMK inhibitor of the present invention is used to define the ROMK-sensitive component of thallium flux. Fluorescence in the presence of test compounds is normalized to control values to provide % fluorescence change. IC50 values represent the concentration of compound that inhibits 50% of the ROMK thallium flux signal.
Measurement: IC50 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 476.52, ALogP 2.27, HBA 8, HBD 0, PSA 93.63, QED 0.53
Compound (SMILES): O=C(Nc1ccc(OC(F)(F)Cl)cc1)c1cnc(N2CC[C@@H](CO)[C@@H](O)C2)c(-c2cncnc2)c1
InChIKey: SQSOTCHJYRODHA-XOBRGWDASA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Cellular Proliferation Assay: Test compounds were resuspended in DMSO at a concentration of 10 mM. A serial three-fold dilution of each compound with DMSO was performed in 384-well plates using the Janus Liquid Dispenser (PerkinElmer). Compound was delivered to the assay plates containing 2500 cells in a 50 μL volume via Acoustic delivery from an ATS-100 (EDC). For Ba/F3-BCR-ABL1-WT cell assays, 2 nL of each compound dilution was transferred to the assay plate for final assay concentrations of 0.4 μM, 0.13 μM, 0.044 μM, 0.015 μM, 0.005 μM, 0.001 μM, 0.00033 μM, 0.00011 μM, 0.000037 μM, 0.000012 μM. For Ba/F3-WT and Ba/F3-BCR-ABL1-T315I cell assays, 50 nL of each compound dilution was transferred to the assay plate for final assay concentrations of 10 μM, 3.33 μM, 1.11 μM, 0.37 μM, 0.12 μM, 0.041 μM, 0.014 μM, 0.0046 μM, 0.0015 μM, 0.00051 μM.Cells were incubated at 37° C. in a humidified environment with 5% car
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 505.91, ALogP 3.14, HBA 8, HBD 3, PSA 120.7, QED 0.42
Compound (SMILES): Nc1ncc(-c2ccccc2)nc1C(=O)Nc1ccccc1N1CCC(N)CC1
InChIKey: JWHUZKCDLIXMSE-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: GSKbeta Assay: Types of GSK-3 assay used to test the selectivity/off target potential compounds of the invention with respect to PKC Î±/Î¸ inhibition activity includes the following: Type 1: The GSK-3 specific peptide used in this assay was derived from the phosphorylation site of glycogen synthase and its sequence is: YRRAAVPPSPSLSRHSSPHQ(S)EDEEE (SEQ ID NO: 1). (S) is pre-phosphorylated as is glycogen synthase in vivo and the three consensus sites for GSK-3 specific phosphorylation are underlined. The buffer used to make up the glycogen synthase peptide and [γ-33P] ATP consisted of MOPS 25 mM, EDTA 0.2 mM, magnesium acetate 10 mM, Tween-20 0.01% and mercaptoethanol 7.5 mM at pH 7.00. The compounds were dissolved in dimethyl sulphoxide (DMSO) to a final concentration of 100 mM. Various concentrations were made up in DMSO and mixed with the substrate (GSK-3 peptide) solution (to a final concentration 20 uM) described in the above section along with rabbit or human GSK-3α and GSK-3β (final concentration 0.5 uM/mL enzyme). The reactions were initiated with the addition of [γ-33P] ATP (500 cpm/pmole) spiked into a mixture of ATP (final concentration of 10 uM). After 30 minutes at room temperature the reaction was terminated by the addition of 10 uL of H3PO4/O.OP/0 Tween-20 (2.5%). A volume (10 uL) of the mixture was spotted onto P-30 phosphocellulose paper (Wallac & Berthold, EG&G Instruments Ltd, Milton Keynes). The paper was washed four times in H3PO4 (0.5%), 2 minutes for each wash, air dried and the radioactive phosphate incorporated into the synthetic glycogen synthase peptide, which binds to the P-30 phosphocellulose paper, was counted in a Wallac microbeta scintillation counter Analysis of Data: Values for IC50 for each inhibitor were calculated by fitting a four-parameter logistic curve to the model: cpm=lower+(upper-lower)/(I+(concentration IC50)).
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 388.48, ALogP 2.91, HBA 6, HBD 3, PSA 110.16, QED 0.63
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#Cc1ccc(N)cn1)c1nc2cccc(Cl)c2c(=O)n1-c1ccccc1
InChIKey: UGJJMNPKJPAKIM-INIZCTEOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 508.97, ALogP 3.96, HBA 9, HBD 3, PSA 137.63, QED 0.31
Compound (SMILES): O=C1NCCc2ccc(OCCCCN3CCCN(c4cccc(Cl)c4Cl)CC3)cc21
InChIKey: HDTSSQFZHKNWGP-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Beta-Arrestin Recruitment (Tango) Assay: Recruitment of β-arrestin to agonist-stimulated D2 receptors was performed using a previously described Tango-type assay (Barnea et al., Proc. Natl. Acad. Sci. USA 105:64 (2008)). Briefly, HTLA cells stably expressing β-arrestin-TEV protease and a tetracycline transactivator-driven luciferase were plated in 15-cm dishes in DMEM containing 10% fetal bovine serum and transfected (via calcium phosphate) with 20 μg of a D2V2-TCS-tTA construct (Barnea et al., Proc. Natl. Acad. Sci. USA 105:64 (2008)). The next day, cells were plated in white, clear-bottom, 384-well plates (Greiner, 10,000 cells/well, 50 μl/well) in DMEM containing 1% dialyzed fetal bovine serum. The following day, the cells were challenged with 10 μl/well of reference agonist (6 μM) or D2 test ligand (6 μM)±reference agonist prepared in HBS, 20 mM HEPES, pH 7.4, 18% DMSO (final ligand concentrations were 1 μM, final DMSO concentration was 3%).
Measurement: EC50 = 6.2 nM (pChEMBL 8.210000038146973)
Descriptors: MW 462.42, ALogP 4.65, HBA 4, HBD 1, PSA 44.81, QED 0.61
Compound (SMILES): Nc1nnc(-c2ccc3[nH]cc(-c4nccc(-c5ccccc5Cl)n4)c3c2)o1
InChIKey: PKFIEHJDQBPPOI-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 15.7 nM (pChEMBL 7.800000190734863)
Descriptors: MW 388.82, ALogP 4.58, HBA 6, HBD 2, PSA 106.51, QED 0.47
Compound (SMILES): CCN(CC)CCCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccccc1)NC(=O)c1ccc(C(=O)OC)cc1)C(=O)N[C@@H](CO)C(=O)OC
InChIKey: ZEQPUKRMOYRHJH-YNUAGZMFSA-N
Target: Chromobox protein homolog 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HC52
Assay: Antagonist activity at recombinant human N-terminal His-tagged CBX8 chromodomain (8 to 61 residues) expressed in Escherichia coli Rosetta BL21(DE3)pLysS using biotin-labeled H3K9me3 measured after 30 mins by AlphaScreen assay
Measurement: IC50 = 4800 nM (pChEMBL 5.320000171661377)
Descriptors: MW 796.96, ALogP 1.5, HBA 11, HBD 6, PSA 221.57, QED 0.07
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3cccc4ccccc34)CC2)C1
InChIKey: HHAUIQVPGMLYMC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In vitro JAK Kinase Assay: The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.)
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 516.61, ALogP 4.2, HBA 7, HBD 1, PSA 106.73, QED 0.38
Compound (SMILES): COc1cc(OC)cc(N(CCN2CCC[C@H]2C)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: CHNWMSQQHAFKHI-FSRHSHDFSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 446.68 nM (pChEMBL 6.349999904632568)
Descriptors: MW 472.59, ALogP 4.67, HBA 8, HBD 0, PSA 68.54, QED 0.37
Compound (SMILES): CCOc1ccc(CC(=O)O)cc1-c1ccc(F)c2c1CN(C(=O)CC(C)c1ccc(F)cc1)CC2
InChIKey: DQJYHQZZSONAHV-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 3.9 nM (pChEMBL 8.40999984741211)
Descriptors: MW 493.55, ALogP 5.74, HBA 3, HBD 1, PSA 66.84, QED 0.43
Compound (SMILES): Cc1nc(C(C)(CC2CC2)NC(=O)c2cc(OCC(F)F)c(C3CC3)cn2)no1
InChIKey: YIATZXPSLJFHJG-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 = 5.8 nM (pChEMBL 8.239999771118164)
Descriptors: MW 406.43, ALogP 3.74, HBA 6, HBD 1, PSA 90.14, QED 0.68
Compound (SMILES): NC1=N[C@](CF)(c2cc(-c3nc4ccc(Cl)cc4o3)ccc2F)C[C@@H](C(F)(F)F)O1
InChIKey: SBFYDEMGEKSXPD-MAUKXSAKSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro BACE Cell-Based Assay: The cell-based assay measures inhibition or reduction of Aβ40 in conditioned medium of test compound treated cells expressing amyloid precursor protein. Cells stably expressing Amyloid Precursor Protein (APP) were plated at a density of 40K cells/well in 96 well plates (Costar). The cells were cultivated for 24 hours at 37° C. and 5% CO2 in DMEM supplemented with 10% FBS. The test compounds were then added to cells in 10-point dose response concentrations with the starting concentration being either 100 uM or 10 uM. The compounds were diluted from stock solutions in DMSO and the final DMSO concentration of the test compounds on cells was 0.1%. After 24 h of incubation with the test compounds the supernatant conditioned media was collected and the Aβ 40 levels were determined using a sandwich ELISA.
Measurement: IC50 = 2810 nM (pChEMBL 5.550000190734863)
Descriptors: MW 445.78, ALogP 5.12, HBA 5, HBD 1, PSA 73.64, QED 0.57
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CCC(NCc2ccccc2F)CC1
InChIKey: UFVCCWUFMPSZKU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 465.51, ALogP 3.74, HBA 7, HBD 3, PSA 121.65, QED 0.44
Compound (SMILES): [2H]C([2H])([2H])Oc1cc(OC([2H])([2H])[2H])cc(N(CC#Cc2ccncc2)c2ccc3ncc(-c4cnn(C(C)C)c4)nc3c2)c1
InChIKey: PWYSFHRYCZIJJM-LIJFRPJRSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 5888.44 nM (pChEMBL 5.230000019073486)
Descriptors: MW 504.59, ALogP 5.68, HBA 8, HBD 0, PSA 78.19, QED 0.26
Compound (SMILES): FC(F)(Br)CN[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: GWATZLTXUAFPCW-MGCOHNPYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 70 nM (pChEMBL 7.159999847412109)
Descriptors: MW 399.24, ALogP 3.21, HBA 5, HBD 2, PSA 70.9, QED 0.66
Compound (SMILES): Cc1ccc(NC(=O)c2cccnn2)cc1-c1ccnc(N2CCOCC2)c1
InChIKey: QFSJWERPFONKIA-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 77.92 nM (pChEMBL 7.110000133514404)
Descriptors: MW 375.43, ALogP 2.94, HBA 6, HBD 1, PSA 80.24, QED 0.76
Compound (SMILES): COc1cc(OC)cc(N(CCCN2C[C@@H](C)N[C@@H](C)C2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: IJENXCZIMOXLKN-OYRHEFFESA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 3548.13 nM (pChEMBL 5.449999809265137)
Descriptors: MW 515.66, ALogP 4.26, HBA 9, HBD 1, PSA 80.57, QED 0.35
Compound (SMILES): COc1ccc(-n2nc(C(C)C)cc2NC(=O)Nc2ccc(Oc3ccnc(Nc4cc(C)c5[nH]ncc5c4)n3)c3ccccc23)cc1
InChIKey: CCRFXSGDDYALRL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: c-Src and Syk Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 uL) is incubated with the test compound (either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL, 2.5 uL each) for 2 hr at RT. The FRET peptides (8 uM, 2.5 uL), and appropriate ATP solutions (2.5 uL, 800 uM for c-Src, and 60 uM ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 639.72, ALogP 8.31, HBA 9, HBD 4, PSA 143.9, QED 0.12
Compound (SMILES): CCCC[C@H](NC(C)=O)C(=O)N[C@H]1CCC(=O)NC[C@@H](C(=O)N[C@@H](Cc2c[nH]c3ccccc23)C(=O)O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](Cc2c[nH]cn2)NC1=O
InChIKey: XHJGHJGQNIFKTO-WNZMCKAXSA-N
Target: Melanocyte-stimulating hormone receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q01726
Assay: cAMP Assay: Accumulation of intracellular cAMP was examined as a measure of the ability of the peptides of the present invention to elicit a functional response in a human melanoma cell line, HBL, that express hMCR-1 (see Kang, L., et al., A selective small molecule agonist of melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice, J. Leuk. Biol. 80:897-904 (2006)) or HEK-293 cells that express hMCR-4. Confluent HBL cells that express hMCR-1 or HEK-293 cells that express recombinant hMCR-4 were detached from culture plates by incubation in enzyme-free cell dissociation buffer. Dispersed cells were suspended in Earle's Balanced Salt Solution containing 10 mM HEPES (pH 7.5), 1 mM MgCl2, 1 mM glutamine, 0.5% albumin and 0.3 mM 3-isobutyl-1-methyl-xanthine (IBMX), a phosphodiesterase inhibitor. The cells were plated in 96-well plates at a density of 0.4Ã¿105 cells per well for HBL cells and 0.5x105 cells per well for HEK-293 cells and pre-incubated for 10 minutes. Cells were exposed for 15 minutes at 37° C. to peptides of the present invention dissolved in DMSO (final DMSO concentration of 1%) at a concentration range of 0.05-5000 nM in a total assay volume of 200 uL. NDP-α-MSH was used as the reference agonist. cAMP levels were determined by an HTRF cAMP cell-based assay system from Cisbio Bioassays utilizing cryptate-labeled anti-cAMP and d2-labeled cAMP, with plates read on a Perkin-Elmer Victor plate reader at 665 and 620 nM. Data analysis was performed by nonlinear regression analysis with Graph-Pad Prism software. Maximum efficacy (Emax) values were determined for each test peptide of the present invention, compared to that achieved by the reference melanocortin agonist NDP-α-MSH.
Measurement: EC50 = 0.107 nM (pChEMBL 9.970000267028809)
Descriptors: MW 997.13, ALogP -1.22, HBA 11, HBD 14, PSA 376.47, QED 0.03
Compound (SMILES): c1cc2c(ncn3nnc(C4CCN(C5CCCC5)CC4)c23)[nH]1
InChIKey: NRQQFRUCNQDBDE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 310.41, ALogP 2.73, HBA 5, HBD 1, PSA 62.11, QED 0.79
Compound (SMILES): C[C@@H](CN1CCC2(CC1)C(=O)NC[C@@H]2c1ccc(F)cc1)NC(=O)c1ccc(F)cc1
InChIKey: ZRRZCDIHCHFDRR-HRAATJIYSA-N
Target: Phospholipase D1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13393
Assay: Inhibition Assay: In one aspect, the compound inhibits PLD activity; that is, a compound can inhibit PLD1 activity and/or PLD2 activity. In a further aspect, the compound inhibits PLD1 response in Calu-1 cells. In a further aspect, the compound inhibits PLD2 response in HEK293gfpPLD2 cells. In a further aspect, the compound inhibits in vitro PLD1 response. In a further aspect, the compound inhibits in vitro PLD2 response.
Measurement: IC50 = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 427.5, ALogP 3.08, HBA 3, HBD 2, PSA 61.44, QED 0.77
Compound (SMILES): O=[SH](=O)Nc1ccc(-c2ccc3ncnc(Nc4cccc5[nH]ncc45)c3c2)cc1
InChIKey: PTBJHZNXVJXIPX-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Kinase Assay: Inhibition of kinase activity by test compound disclosed herein was estimated by quantifying the amount of [33P] incorporation of substrate in the presence of test compound. Standard assay conditions were 2 ng of recombinant C-Raf or 5 ng of recombinant B-Raf or 5 ng of recombinant B-Raf (V600E) kinase (Upstate Biotechnology) with 500 ng MEK1(K97R) in assay buffer {8 μM ATP, 0.5 μCi [33P]ATP (specific activity 3000 Ci/mmol, PerkinElmer), 50 mM Tris/HCl (pH7.5), and 1 mM EGTA, 1 mM Na3VO4, 1% 2-mercaptoethanol, 0.1% Brij 35, and 0.2 mg/ml BSA} at a final volume of 25 μL. Reactions were incubated at 30° C. for 30 min and stopped by adding 3% phosphoric acid, harvested onto a 96-well GF/B UniFilter (PerkinElmer) using a unifilter harvester (PerkinElmer), and counted with a TopCount microplate scintillation counter (PerkinElmer). The IC50 values of inhibitors were determined after carrying out assays at 3-fold serially diluted concentrations of each compound in duplication.
Measurement: IC50 = 677.1 nM (pChEMBL 6.170000076293945)
Descriptors: MW 416.47, ALogP 3.86, HBA 6, HBD 4, PSA 112.66, QED 0.32
Compound (SMILES): Cc1ccc(F)c2nc([C@H](C)Nc3ncnc(N)c3C#Cc3cnccn3)n(-c3ccccc3)c(=O)c12
InChIKey: UGDKXZUULFSWDZ-KRWDZBQOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 492.52, ALogP 3.57, HBA 9, HBD 2, PSA 124.5, QED 0.37
Compound (SMILES): CS(=O)(=O)c1ccc(CNC[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: JYPQLENCUJFZLR-JCNLHEQBSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 710 nM (pChEMBL 6.150000095367432)
Descriptors: MW 438.56, ALogP 3.07, HBA 7, HBD 2, PSA 105.04, QED 0.48
Compound (SMILES): CCOc1ccccc1N1CCCN(CCCCOc2ccc3ccc(=O)[nH]c3c2)CC1
InChIKey: UAILOOIRJOOOAV-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Beta-Arrestin Recruitment (Tango) Assay: Recruitment of β-arrestin to agonist-stimulated D2 receptors was performed using a previously described Tango-type assay (Barnea et al., Proc. Natl. Acad. Sci. USA 105:64 (2008)). Briefly, HTLA cells stably expressing β-arrestin-TEV protease and a tetracycline transactivator-driven luciferase were plated in 15-cm dishes in DMEM containing 10% fetal bovine serum and transfected (via calcium phosphate) with 20 μg of a D2V2-TCS-tTA construct (Barnea et al., Proc. Natl. Acad. Sci. USA 105:64 (2008)). The next day, cells were plated in white, clear-bottom, 384-well plates (Greiner, 10,000 cells/well, 50 μl/well) in DMEM containing 1% dialyzed fetal bovine serum. The following day, the cells were challenged with 10 μl/well of reference agonist (6 μM) or D2 test ligand (6 μM)±reference agonist prepared in HBS, 20 mM HEPES, pH 7.4, 18% DMSO (final ligand concentrations were 1 μM, final DMSO concentration was 3%).
Measurement: EC50 = 4.8 nM (pChEMBL 8.319999694824219)
Descriptors: MW 435.57, ALogP 4.3, HBA 5, HBD 1, PSA 57.8, QED 0.51
Compound (SMILES): COc1ccc(-c2ccc3ncnc(-c4cccc(C(=O)N5CCN(C(C)=O)CC5)c4)c3c2)cc1OC
InChIKey: NADSMCCUYCEYHM-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Inhibition of PI3K-delta (unknown origin) by KinaseGlo assay
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 496.57, ALogP 4.29, HBA 6, HBD 0, PSA 84.86, QED 0.41
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(-c3ccc4oc(N)nc4c3)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: NAFFBOIJDJRKKK-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of recombinant human C-terminal His6-tagged p110gamma expressed in insect cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 528.58, ALogP 4.77, HBA 10, HBD 2, PSA 143.67, QED 0.33
Compound (SMILES): C=CC(=O)Nc1cccc(Oc2nc(Nc3ccc(OC)c(OC)c3OC)ncc2Cl)c1
InChIKey: KKXHOHZIJWZIMH-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Omnia Assay: Briefly, 10x stocks of EGFR-WT (PV3872) from Invitrogen and EGFR-T790M/L858R (40350) from BPS Bioscience, San Diego, Calif., 1.13x ATP (AS001A) and appropriate Tyr-Sox conjugated peptide substrates (KCZ1001) were prepared in 1x kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10x stock, KB002A) and 0.2 mM DTT (DS001A). 5 uL of each enzyme were pre-incubated in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.) for 30 min. at 25° C. with a 0.5 uL volume of 50% DMSO and serially diluted compounds prepared in 50% DMSO. Kinase reactions were started with the addition of 45 uL of the ATP/Tyr-Sox peptide substrate mix and monitored every 71 seconds for 60 minutes at λex360/λem485 in a Synergy4 plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 456.89, ALogP 4.82, HBA 8, HBD 2, PSA 103.83, QED 0.44
Compound (SMILES): COc1ccc(OCc2cn(-c3ccc(S(N)(=O)=O)cc3)nn2)cc1
InChIKey: WZONQFASBANDEJ-UHFFFAOYSA-N
Target: Carbonic anhydrase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16790
Assay: Inhibition of human carbonic anhydrase 9 pre-incubated for 15 mins by stopped flow carbon dioxide hydrase assay
Measurement: Ki = 14.3 nM (pChEMBL 7.840000152587891)
Descriptors: MW 360.39, ALogP 1.5, HBA 7, HBD 1, PSA 109.33, QED 0.71
Compound (SMILES): Cc1n[nH]c(C)c1S(=O)(=O)N1CCC(Oc2ccc3ccccc3c2)CC1
InChIKey: AXYFHHUNIHMJGQ-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Biological Assay: The ability of a compound to block the intracellular release of calcium mediated by PK1 in RBL2H3 cells expressing human PKR1 receptors is determined as a measure of the compound's antagonist activity in vitro.Approximately 10,000 cells per assay well are seeded in normal culture medium in a 384 well plate (Corning). Twenty-four hours after seeding, the cells are loaded with a calcium sensitive fluorescent dye by replacing the culture medium with assay buffer (1× Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH 7.4) containing 1 mM probenecid and 1× Calcium 5 Reagent (Molecular Devices). Cells are incubated at 37° C. for 1 hour to allow for dye uptake.To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 μM (diluted in assay buffer) are added to the assay wells and allowed to incubate for 10 minutes prior to stimulation with PK1. After incubation with test compounds the assay plate is placed in a FLIPR Tetra (Molecular Devices) and PK1 (diluted in assay buffer) is added at the determined EC80 concentration (final). Ligand-dependent changes in intracellular calcium levels are determined by measuring changes in fluorescence of the dye at 525 nM following to excitation at 485 nM.
Measurement: IC50 = 440 nM (pChEMBL 6.360000133514404)
Descriptors: MW 385.49, ALogP 3.41, HBA 4, HBD 1, PSA 75.29, QED 0.75
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3cc(C(F)(F)F)ncc3F)CC2)C1
InChIKey: JDKXQCYZSUBGIZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In vitro JAK Kinase Assay: The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.)
Measurement: IC50 = 12.5 nM (pChEMBL 7.900000095367432)
Descriptors: MW 553.52, ALogP 3.6, HBA 8, HBD 1, PSA 119.62, QED 0.38
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COCc4ccccc4C)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: YWYRCGRSOXUMBY-SROIDHQSSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Binding Assay: Binding assay of certain compounds of this invention to the opioid receptor was determine using radioligand binding assay (screening and dose-displacement).
Measurement: Ki = 0.07 nM (pChEMBL 10.149999618530273)
Descriptors: MW 515.69, ALogP 5.44, HBA 5, HBD 0, PSA 40.16, QED 0.47
Compound (SMILES): Cc1c(-c2c(Cl)cc(C(N)=O)c3[nH]c4c(c23)CC[C@@H](C(C)(C)O)C4)cccc1-n1c(=O)c2cccc(F)c2n(C)c1=O
InChIKey: KABDJJRRZYJNRL-MRXNPFEDSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition of BTK in human whole blood assessed as suppression of BCR/anti-IgM/IgG-stimulated CD69 surface expression after 18 hrs with agitation by FITC-staining based FACS analysis
Measurement: IC50 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 589.07, ALogP 4.91, HBA 6, HBD 3, PSA 123.11, QED 0.28