lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN(C(=O)[C@H]1C[C@]2(COC(=O)N2)C1)[C@H]1C[C@@H](Cc2ccccc2)C1
InChIKey: FKXACVTZYYOQAZ-YIGXGSLSSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Activity Assay: The assay used to measure the in vitro activity of MGL is adapted from the assay used for another serine hydrolase (FAAH) described in Wilson et al., 2003 (A high-throughput-compatible assay for determining the activity of fatty acid amide hydrolase. Wilson S J, Lovenberg T W, Barbier A J. Anal Biochem. 2003 Jul. 15; 318(2):270-5.). The assay consists of combining endogenously expressed MGL from HeLa cells with test compounds, adding [glycerol-1,3-3H]-oleoyl glycerol, incubating for one hour, and then measuring the amount of cleaved [1,3-3H]-glycerol that passes through an activated carbon filter. The amount of cleaved, tritiated glycerol passing through the carbon filter is proportional to the activity of the MGL enzyme in a particular well/test condition.
Measurement: IC50 = 7.8 nM (pChEMBL 8.109999656677246)
Descriptors: MW 328.41, ALogP 2.35, HBA 3, HBD 1, PSA 58.64, QED 0.92
Compound (SMILES): CC(C)S(=O)(=O)N1CCN(c2nc3nc(-c4noc(=O)[nH]4)nc(N[C@H](C)C4CCC4)c3n2C[C@H]2CC[C@H](C)CC2)[C@H](C)C1
InChIKey: KHJXRSXKSRWQAV-XRXFAXGQSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 10.08 nM (pChEMBL 8)
Descriptors: MW 615.81, ALogP 3.85, HBA 11, HBD 2, PSA 155.14, QED 0.36
Compound (SMILES): CC(C)c1nnc(Cn2ncc3ncc(-c4ccc(F)c(C(F)F)c4)cc32)o1
InChIKey: BYWYYVGUXZBASY-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: Effects of Test Articles on Cloned Human NR1/GuN2B Ion Channels Expressed in Mammalian Cells: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty-four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed, and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2 (standard assay) or 1.5 mM MgCl2 (HTS assay), 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2), 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC40 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 387.37, ALogP 4.73, HBA 6, HBD 0, PSA 69.63, QED 0.5
Compound (SMILES): COC(=O)N1Cc2cc(Cl)ccc2-n2c(nnc2C2CC3(C2)CN(c2ncc(F)cn2)C3)C1
InChIKey: SJIHKOVAXYDWLP-UHFFFAOYSA-N
Target: Vasopressin V1a receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P37288
Assay: Vasopressin V1A Receptor Antagonist Assay: The purpose of this assay was to determine the inhibitory effect of synthesized compounds on the Vasopressin V1a receptor. The assay as performed in Chinese Hamster Ovary (CHO) cells expressing the human Arginine Vasopressin Receptor 1a (AVPR1a). Arginine Vasopressin (AVP) evokes an increase in intracellular calcium in CHO-AVPR1a cells which is measured in a fluorescence assay on the FLIPRTETRA using calcium sensitive dyes. Test compounds were assessed for their ability to affect the magnitude of the response to AVP, with antagonists showing a concentration-dependent reduction in the AVP-mediated fluorescence. Compounds were tested in duplicate in a 10-point, 1:3 dilution series starting at a nominal concentration of 3 μM in the assay.CHO-AVPR1a cells were maintained in routine culture in T175 Flasks at 37° C., 5% CO2. The growth medium consists of Ham's F12 media supplemented with 10% v/v fetal bovine serum, 1× non-essential amino acids, and 0.4 mg/ml Geneticin G418.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 469.91, ALogP 3.32, HBA 8, HBD 0, PSA 89.27, QED 0.57
Compound (SMILES): CN(C)c1cnc2ccc(-c3n[nH]c4nc(N5[C@H]6CC[C@@H]5[C@@H](F)[C@@H](N)C6)c(CO)nc34)c(Cl)c2n1
InChIKey: JABIEEOYCYLUIX-LFNKTWDUSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Biochemical Assay: SHP2 activity was monitored by measuring the conversion of the surrogate substrate 6,8-difluoromethylumbelliferyl phosphate (DiFMUP) to the fluorescent product, 6,8-difluoromethylumbelliferone (DiFMU).SHP2 was pre-incubated with test compounds and the activating peptide pIRS1 (H2N-LN(pY)IDLDLV-(PEG)8-LST(pY)ASINFQK-amide) for 30 min, prior to addition of the 6,8-difluoromethylumbelliferyl phosphate (DiFMUP), (Thermo Fisher D6567). Final assay concentrations were 10 μM SHP2, 0.25 μM pIRS1 peptide, 50 μM DiFMUP, 25 mM Bis-Tris propane, pH 7.0, 150 mM NaCl, 0.05% (v/v) Tween-20, 0.5 mM TCEP and 5% (v/v) DMSO. Rates of reaction were then measured over 30 min by monitoring fluorescence on a BMG Pherastar reader at excitation 360 nm/emission 450 nm. IC50 values were calculated from the normalized dose-response plots using four parameter logistic curve fit.
Measurement: IC50 = 5.3 nM (pChEMBL 8.279999732971191)
Descriptors: MW 497.97, ALogP 2.58, HBA 9, HBD 3, PSA 132.97, QED 0.39
Compound (SMILES): O=C([C@@H]1CC[C@H](NC2(C(F)(F)F)CC2)CO1)N1Cc2cccnc2Nc2ccc(N3CCOCC3)cc21
InChIKey: ZHDAOEDYYWTTDM-AVRDEDQJSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Enzymatic Assay: Each test compound (10 mM stock in DMSO) is diluted in DMSO to make a 10-point, 3-fold dilution series. 125 nL of each dilution or DMSO alone is dispensed to a 384-well Greiner Lumitrac 200 assay plate using an Echo® Liquid Handler. To each well of the plate is added 20 uL of enzyme in assay buffer or assay buffer alone. Assay buffer consists of 50 mM sodium phosphate, pH 7.0, 50 mM magnesium chloride, 50 mM sodium chloride, and 0.01% (w/v) bovine serum albumin. When present, the R132H mutant IDH1 enzyme is at a working concentration of 1.875 nM (final concentration in assay of 1.5 nM). The assay plate is allowed to incubate for 30 minutes at room temperature and 5 uL of 5× substrate mixture (2.5 uM nicotinamide adenine dinucleotide phosphate, 100 uM adenosine diphosphate, 7.5 mM glyceraldehyde-3-phosphate, 7.5 ug/mL of spinach glyceraldehyde-3-phosphate dehydrogenase, 25 nM phosphoglycerate kinase, and 5 mM alpha-ketoglutarate in assay buffer) is added to all wells. The reaction plate is incubated for 60 minutes followed by addition of 25 uL of Promega Kinase-GLO reagent to all wells and 10-minute incubation.Luminescence is measured using a PerkinElmer Envision plate reader. The percent activity of each dilution is determined as the ratio of background corrected signal to the background corrected signal of wells receiving only DMSO. IC50 values are determined by fitting percent activity data to a four-parameter logistic dose response equation.
Measurement: IC50 = 57 nM (pChEMBL 7.239999771118164)
Descriptors: MW 517.55, ALogP 3.74, HBA 7, HBD 2, PSA 78.96, QED 0.64
Compound (SMILES): CCCn1cc(-n2c(Cl)c(Sc3cccc(C(=O)O)c3)c3ccc(Cl)c(F)c32)cn1
InChIKey: CHFTWRTZSPUBLH-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Human Autotaxin Assay: ATX activity is assayed in concentrated conditioned media from Hep3B human hepatocellular carcinoma cells by measuring the amount of choline released from the substrate, lysophosphatidylcholine (LPC) as it is cleaved to LPA. Conditioned media is collected from confluent Hep3B cells and concentrated 20-fold using Centriprep-30 filter devices (Millipore). To assay for autotaxin inhibition, 10-20 μL of the concentrated conditioned media is incubated with 2.5 μL of a test compound in DMSO and 72.5-82.5 μL lyso-PLD buffer (100 mM Tris pH 9, 500 mM NaCl, 5 mM MgCl2, 5 mM CaCl2, 0.05% Triton X-100 in the presence or absence of 0.2% fatty-acid-free human serum albumin) for 15 min at 37° C. After the 15 min incubation, 5 ul of 2 mM LPC (14:0; Avanti Polar Lipids Cat #855575C) diluted in lyso-PLD buffer is added for a final concentration of 100 uM and the incubation continues for 1.5-3 hours at 37° C. 100 μl of a color mix containing 4.5 mM 4-aminoantipyrine, 2.7 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine, 21 units/ml horseradish peroxidase and 3 units/ml choline oxidase in 50 mM Tris, pH 8, 4.5 mM MgCl2 is added and the incubation continued for 15 minutes at room temperature before reading the absorbance at 555 nm.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 464.35, ALogP 6.53, HBA 5, HBD 1, PSA 60.05, QED 0.35
Compound (SMILES): CC(=O)N[C@@H](C)CO[C@H]1C[C@H](COc2ncnc(OCc3ccc(F)cc3)c2Cl)C1
InChIKey: WCVWWMDCDGFDLB-OWQGQXMQSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Inhibitory Activity on Human ACC1 and the ACC2: Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H]−+Intensity of [AcCoA-H]−) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 437.9, ALogP 3.55, HBA 6, HBD 1, PSA 82.57, QED 0.61
Compound (SMILES): CC(=O)c1c(C)c2cnc(N[C@@H]3CCOC[C@H]3O)nc2n([C@H]2CC[C@H](N)CC2)c1=O
InChIKey: JHSKWDLRXRYFOQ-MUIFIZLQSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24385
Assay: Chelation-Enhanced Fluorescence (CHEF) Assay: CDK4/Cyclin D1 and CDK6/Cylcin D3: The Chelation-Enhanced Fluorescence (CHEF) monitors the phosphorylation state in real time where the level of fluorescence is directly proportional to the amount of phosphorylated substrate. CHEF exploits a synthetic α-amino acid with a side chain bearing an 8-hydroxyquinoline derivative (sulfonamido-oxine, Sox) which, upon coordination to Mg(II), relays information on the phosphorylation state of a proximal serine, threonine or tyrosine residue in peptide-based kinase substrates. Phosphorylation of a specific peptide (Catalog # AQT0258) from AssayQuant Technologies results in an increase in fluorescence at the excitation and emission wavelengths of 360 nm Ex/485 nm Em.Examples along with DMSO (negative) and Palbociclib (positive) controls were added to 384-well plates at 100-fold their final concentrations followed by the addition of 10 nM CDK4/Cyclin D1 (LJIC-2007F1) or 10 nM CDK6/Cyclin D3 (LJIC-2009H2) for a 20 minute pre-incubation in assay buffer containing 40 mM HEPES, 1 mM Dithiothreito (DTT, 10 mM MgCl2, 1% Glycerol, 0.1% BSA. Enzyme reactions were initiated by the addition of AssayQuant Technologies peptide and ATP substrates (10 μM CHEF peptide (Catalog # AQT0258), 2 mM ATP) and allowed to proceed for 2 hours followed by fluorescence read of the reaction. Ki determinations were made from a plot of the fractional velocity as a function of inhibitor concentration fit to the Morrison equation with the enzyme concentration as a variable.
Measurement: Ki = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 415.49, ALogP 1.31, HBA 9, HBD 3, PSA 132.36, QED 0.64
Compound (SMILES): COc1ccccc1C(Cn1c(=O)n(C2(C(=O)O)CC2)c(=O)c2c(C)c(C#N)sc21)OC1CCOCC1
InChIKey: AZCJQLYZOQFTBF-UHFFFAOYSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: In Vitro Acetyl-CoA Carboxylase (ACC) Inhibition Assay: An exemplary procedure for the in vitro ACC inhibition assay, which can be used to determine the inhibitory action of compounds of the invention toward either ACC1 or ACC2, follows. The ADP-Glo Kinase Assay kit from Promega was used. The ADP-Glo Kinase Assay is a luminescent ADP detection assay to measure enzymatic activity by quantifying the amount of ADP produced during an enzyme reaction. The assay is performed in two steps; first, after the enzyme reaction, an equal volume of ADP-Glo Reagent is added to terminate the reaction and deplete the remaining ATP. Second, the Kinase Detection Reagent is added to simultaneously convert ADP to ATP and allow the newly synthesized ATP to be measured using a luciferase/luciferin reaction. Luminescence can be correlated to ADP concentrations by using an ATP-to-ADP conversion curve. The detailed procedure is as follows. 50 μL of the compound being tested (600 uM in DMSO) was added to a 384-well dilution plate. The compound was diluted 1:3 in succession in DMSO for each row for 11 wells. 0.5 μL ACC2 working solution was added to 384-well white Optiplate assay plate. 0.5 μL diluted compound solution in each column from step 2 to assay plate, each row containing 2 replicates. For the last 2 rows, add 0.5 μL negative control (DMSO) in one row and 0.5 μL positive control (compound 1-97) in the other. The plates were incubated at room temperature for 15 minutes. 5 μL substrate working solution was added to each well to initiate reaction. Final ACC2 reaction concentrations consist of: 5 nM ACC2, 20 μM ATP, 20 μM acetyl-CoA, 12 mM NaHCO3, 0.01% Brij35, 2 mM DTT, 5% DMSO, test compound concentrations: 30 μM, 10 μM, 3.33 μM, 1.11 μM, 0.37 μM, 0.123 μM, 0.0411 μM, 0.0137 μM, 0.00457 μM, 0.00152 μM, and 0.00051 μM. Plates were incubated at room temperature for 60 minutes. 10 μL ADP glo reagent was added. Plates were incubated at room temperature for 40 minutes. 20 μL kinase detection reagent was added. Plates were incubated at room temperature for 40 minutes, then read on a Perkin Elmer EnVision 2104 plate reader for luminescence as Relative Light Units (RLU).Data for each concentration, as well as the positive and negative controls were averaged, and the standard deviation calculated. Percent inhibition was calculated by the formula: 100×(average negative control-compound)/(average negative control-average positive control). The IC50 for each compound was calculated by fitting the data with a non-linear regress ion equation: Y=Bottom+(Top−Bottom)/(1+10((Log IC50−X* HillSlope)), where X is the log of compound concentration and Y is percent inhibition.As shown in the following table, tested compounds of t
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 525.58, ALogP 2.92, HBA 10, HBD 1, PSA 132.78, QED 0.47
Compound (SMILES): Nc1nn2cc(F)cnc2c1C(=O)Nc1cnccc1N1CCC(S(=O)(=O)C2COC2)CC1
InChIKey: JAWDZHWBXQDRQM-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM). The reaction was stopped after 24 hours by the addition of 30 μL 0.1M phosphoric acid containing 2 mM ATP. A multiscreen phosphocellulose filter 96-well plate (Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2M phosphoric acid prior to the addition of 45 μL of the stopped assay mixture. The plate was washed with 5×200 μL 0.2M phosphoric acid. After drying, 100 μL Optiphase SuperMix liquid scintillation cocktail (Perkin Elmer) was added to the well prior to scintillation counting (1450 Microbeta Liquid Scintillation Counter, Wallac).
Measurement: Ki = 505 nM (pChEMBL 6.300000190734863)
Descriptors: MW 475.51, ALogP 0.88, HBA 10, HBD 2, PSA 144.81, QED 0.55
Compound (SMILES): N#CC1(COc2nc(C(=O)N3CCC(F)C3)cc(N3CCC(c4n[nH]c5ncccc45)CC3)n2)CC1
InChIKey: JFXCULKPPZAXKE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Enzyme Assay: AXL enzyme inhibition (% inhibition, Kiapp and Ki values) by small molecule inhibitors was evaluated using a fluorescence-based microfluidic mobility shift assay. AXL catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-30 (5-FAM-KKKKEEIYFFF-CONH2, CPC Scientific, Sunnyvale, Calif.). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Human wild-type receptor tyrosine kinase protein Axl comprising residues 505-811 was produced in-house using the baculoviral expression vector system that incorporated a hexahistidine affinity tag into the protein (LJIC-1916B1.1). The enzyme was preactivated by auto-phosphorylation of 34 uM non-activated enzyme in the presence of 2 mM ATP, 4 mM MgCl2, 50 mM NaCl and 1 mM TCEP in 20 mM HEPES, pH 7.3 at 4° C. for 30 minutes. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 10 mM MgCl2, 1 mM DTT, 120 μM ATP (ATP Km=70.4 μM), 0.01% Tween-20, 3 μM FL-Peptide-30, and 0.5 nM phosphorylated AXL enzyme in 100 mM HEPES buffer at pH 7.3. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 30 minutes at 25° C. by the addition of 50 μL of 200 mM EDTA, pH 7.5. The Ki values were determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable. (See, Morrison, J. F. (1969) Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors, Biochimica et biophysica acta 185, 269-286; and Murphy, D. J. (2004) Determination of accurate KI values for tight-binding enzyme inhibitors: an in silico study of experimental error and assay design, Analytical biochemistry 327, 61-67.)
Measurement: Ki = 19.4 nM (pChEMBL 7.710000038146973)
Descriptors: MW 490.54, ALogP 3, HBA 8, HBD 1, PSA 123.92, QED 0.56
Compound (SMILES): Cc1cc(F)c(C(=O)Nc2cccc(-c3nnnn3C(C)C)n2)cc1-c1cccnc1
InChIKey: CNMBQIRCFFMULX-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: HTRF KinEASE Assay: The ability (IC50) of compounds to inhibit ASK1 kinase activity was determined by HTRF® KinEASE Assay System. ASK1 was purchased from Thermofisher (Catalogue #PV4011), ATP was purchased from Sigma (Catalogue #A7699), HTRF® KinEASE Assay System was obtained from Cisbio (Bedford, Mass.). ½ Area plate was purchased from Perkin Elmer (Catalogue ##6005560). HTRF® KinEASE-STK is a generic method for measuring serine/threonine kinase activities using a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. The IC50 value for each compound was determined in the presence of compound (various concentration from 0 to 10 μM) and a fixed amount of ATP and peptide substrates. The test compound, 1 uM STK3 peptide substrate, and 5 nM of ASK1 kinase are incubated with kinase reaction buffer containing 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1 mM EGTA for 30 minutes. 100 uM ATP is added to start kinase reaction and incubated for 3 hours. The STK3-antibody labeled with Eu3+-Cryptate and 125 nM streptavidin-XL665 are mixed in a single addition with stop reagents provided by the Cisbio kit used to stop the kinase reaction. Fluorescence is detected using an Envision Multilabeled 2014 reader from PerkinElmer. The Fluorescence is measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm is calculated for each well.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 417.45, ALogP 4.08, HBA 7, HBD 1, PSA 98.48, QED 0.53
Compound (SMILES): Cc1nsc(-c2c(NC(=O)C3=C(C(=O)O)CCCC3)sc3c2CCCC3)n1
InChIKey: NPBRYESWINAZTL-UHFFFAOYSA-N
Target: Fatty acid-binding protein, adipocyte (SINGLE PROTEIN), Homo sapiens
UniProt: P15090
Assay: Time Resolved-Fluorescence Energy Transfer (TR-FRET) Assay: Compounds were profiled for activity against human FABP4 (huFABP4) and/or human FABP5 (huFABP5) in Terbium (Tb) time resolved-fluorescence energy transfer (TR-FRET) assays monitoring the direct binding of Bodipy labeled fatty acid to His6 tagged FABP proteins (huFABP4 was expressed in house in E. Coli and purified, huFABP5 was purchased from Cayman Chemical Co., cat.no. 10010364), bound to Terbium labeled anti His6 tag antibody. Assay read-outs reflected energy transfer, upon binding of the ligand to the FABP protein, from the Terbium donor molecule to the acceptor Bodipy moiety. Final ligand concentration (125 nM) approximated the Kd for each protein.Stock DMSO solutions (1.8 mM) of compounds were serially diluted 3-fold for ten concentrations with 100% DMSO (50 μM to 0.003 μM final compound concentration). 1 μl of these compound dilutions and 1 μl of Bodipy labeled fatty acid 4.5 μM in 100% DMSO (Bodipy FL C11, cat. no. D3862, Invitrogen) were sequentially pipetted in wells of 384-well black polypropylene plates (Thermo Matrix cat. no. 4344). FABP4 or FABP5 protein was then added (28 μl of 64 nM protein in 25 mM Tris pH 7.5, 0.4 mg/ml γ-globulin, 1 mM DTT, 0.012% NP40, final protein concentration: 50 nM). Assay blanks contained ligand, but no protein. Neutral controls contained ligand, but no compound. After adding the detection reagent (Tb antiHis6 antibody, Columbia Biosciences, TB-110, 6 μl of a 24 nM Ab solution in 25 mM Tris pH 7.5, 0.4 mg/ml γ-globulin, final Tb antiHis6 Ab concentration: 4 nM), plates were spun one minute at 1000 rpm. Following an incubation at room temperature with shaking for 30 minutes, plates were read using an Envision reader (Perkin Elmer, Extinction wavelength: 340 nm, Emission: 490 nm and 520 nm, time delay: 100 μs; time window: 200 μs, 50 flashes).Final assay conditions were: 50 nM FABP protein, 125 nM Bodipy labeled fatty acid, 0.009% (vol/vol) NP40, 5.5% (vol/vol) DMSO in a total final assay volume of 36 μl. The assay was performed in triplicate.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 403.53, ALogP 4.35, HBA 6, HBD 2, PSA 92.18, QED 0.79
Compound (SMILES): COc1ccccc1-c1cc(F)cc2cnc(Nc3ccc(C4CCNCC4)c4c3OCC4)nc12
InChIKey: QCJIOPQYKRWUJO-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Enzyme Activity Inhibition IC50 Evaluation : Buffer preparation: 50 mM HEPES, pH 7.5, 0.00015% Brij-35.The compound was configured as a concentration gradient in 100% DMSO and added to a 384-well plate with a final DMSO concentration of 2%.1. ALK enzyme (purchased from Carna Biosciences, Inc.) was diluted to the optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 0.00015% Brij-35, 2 mM DTT, which was transferred to a 384-well plate and incubated with the compound for a certain time.2. The substrate was diluted to the optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 0.00015% Brij-35, mM MgCl2, adenosine triphosphate under Km, which was added to the 384-well plate to initiate the reaction and reacted at 28° C. for 1 hour.3. The conversion rate was read by Caliper Reader, the calculated inhibition rate is the average of the two tests.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 470.55, ALogP 5.59, HBA 6, HBD 2, PSA 68.3, QED 0.4
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc4[nH]c(CN5CCC(O)CC5)cc4c3N(C)C2=O)c1F
InChIKey: WHYRSPOYURSJPX-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 487.51, ALogP 3.39, HBA 6, HBD 2, PSA 94.16, QED 0.57
Compound (SMILES): Cc1cc(N2CCOCC2)cc(Cl)c1C(=O)n1nc(-c2ccc(C(=O)O)cc2F)c2ncccc21
InChIKey: XQLORDGAAKKHJV-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biological Assays: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was expressed in SF9 cells using a baculovirus expression system. The RORγ-LBD protein was purified by glutathione sepharose chromatography. Separately, SF9 cells not expressing any recombinant protein were lysed and the lysate was added to the purified RORγ-LBD at 0.25 μl lysate (from 10,000 SF9 cells)/nM purified protein. The mixture was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT) to obtain RORγ-LBD final concentration of 3 nM in 384-well assay plate.Compounds to be tested were injected to the assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, Calif.).A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-CPSSHSSLTERHKILHRLLQEGSPS) (SEQ ID NO:2) was prepared in assay buffer and added to each well (100 nM final concentration). A solution of Europium tagged anti-HIS antibody (1.25 nM final concentration) and APC conjugated streptavidin (8 nM final concentration) were also added to each well.The final assay mixture was incubated overnight at 4° C., and the fluorescence signal was measured on an Envision plate reader: (Excitation filter=340 nm; APC emission=665 nm; Europium emission=615 nm; dichroic mirror=D400/D630; delay time=100 μs, integration time=200 μs). IC50 values for test compounds were calculated from the quotient of the fluorescence signal at 665 nm divided by the fluorescence signal at 615 nm.
Measurement: IC50 = 6866 nM (pChEMBL 5.159999847412109)
Descriptors: MW 494.91, ALogP 4.42, HBA 7, HBD 1, PSA 97.55, QED 0.45
Compound (SMILES): CN(Cc1ccccc1-c1cccnc1)C(=O)c1ccc(Nc2nc3c(C4=CCN(C(=O)CCC(F)(F)F)CC4)cccn3n2)cc1
InChIKey: WZOMMIYEJLUPCD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 1.94 nM (pChEMBL 8.710000038146973)
Descriptors: MW 639.68, ALogP 6.77, HBA 7, HBD 1, PSA 95.73, QED 0.19
Compound (SMILES): C[C@H](Nc1nc(N)nc(N)c1C#N)c1nc2cccc(Cl)c2c(=O)n1C[C@@H]1CCCNC1
InChIKey: HSKOHCUTBYTMMJ-NWDGAFQWSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: a time-resolved fluorescence resonance energy transfer (TR-FRET) assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2×Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. The compounds of Table 1 and Compound X, ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 500 μs read window).The results were normalized based on positive (1 pM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3K α, β, δ, and γ were calculated from the fit of the dose-response curves to a four-parameter equation. These assays generally produced results within 3-fold of the reported mean.
Measurement: IC50 = 88 nM (pChEMBL 7.059999942779541)
Descriptors: MW 453.94, ALogP 2.05, HBA 10, HBD 4, PSA 160.56, QED 0.45
Compound (SMILES): C[C@@H](c1ccc(F)cc1)n1nc(C#N)c2c(=O)[nH]c([C@H]3CC[C@@H]3c3ncccn3)nc21
InChIKey: HAWIPJVSLZKKOE-RCBQFDQVSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Fluorescence Polarization Assay: Human PDE9 (PDE9A2, GenBank Accession No. NM_001001567), full length with N-terminal GST tag, was purchased from BPS Bioscience. The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product #R8139). IMAP® technology has been applied previously to phosphodiesterase assays. Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 μL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate.
Measurement: Ki = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 415.43, ALogP 3.19, HBA 7, HBD 1, PSA 113.14, QED 0.55
Compound (SMILES): CN(C)C1CCN(CCn2nc(-c3[nH]c4ncccc4c3Cl)c3c(N)ncnc32)CC1
InChIKey: BYLSDOKUWICZPL-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Time-Resolved Fluorescence Transfer (FRET) Assay: RET and KDR: Kinase activity was detected using CisBio HTRF kinEASE kit based on time-resolved fluorescence transfer (FRET). The assay was performed in 384-well white plates (Corning #3574) in a reaction volume of 10 μL containing 1× CisBio enzymatic buffer supplemented with a final concentration of 5 mM MgCl2, 1 mM DTT, 10 nM SEB and 0.01% Triton X100 for RET. The same buffer was used for the KDR biochemical assay with the addition of 2 mM MnCl2.Inhibitors were pre-incubated in the plate for 15 mins with 5 μL kinase and assay buffer at the following concentrations; 13 pM RET (Carna Biosciences; 08-159) and 150 pM KDR (Millipore; 14-630). The reaction was initiated by the addition of 5 μL ATP and substrate at 2× final reaction concentrations. For RET, this was 18 pM and 2 μM; for KDR, this was 16 μM and 1 μM, respectively. Reactions were performed at ATP Km for each target. The assay was allowed to proceed at room temperature for 20 mins before terminating with the addition of 10 μL HTRF detection buffer containing EDTA supplemented with TK-antibody labelled with Eu3+-Cryptate (1:100 dilution) and streptavidin-XL665 (128 nM). Following incubation at room temperature for 1 hour, FRET signal was measured using the Pherastar FS Microplate Reader.
Measurement: IC50 = 187 nM (pChEMBL 6.730000019073486)
Descriptors: MW 439.96, ALogP 2.63, HBA 8, HBD 2, PSA 104.78, QED 0.49
Compound (SMILES): N#Cc1cccc(F)c1-c1cc(-n2cc(F)c(N3CCC(O)C3)n2)c2c(n1)CNC2=O
InChIKey: OCCMXLNZRHDUJP-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Caliper Assay: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
Measurement: Ki = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 422.4, ALogP 1.9, HBA 7, HBD 2, PSA 107.07, QED 0.67
Compound (SMILES): Cc1nn(C)cc1[C@@H]1CN(C)C[C@H]1NC(=O)C(C)(C)COc1ncccc1C1CC1
InChIKey: XKSUGUCDWYBSAZ-VQTJNVASSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: Inhibition of Forskolin Stimulated cAMP in Cells Overexpressing SSTR4: This cell-based assay measures the ability of compounds to inhibit forskolin stimulated cAMP in CHO-K1 cells overexpressing SSTR4. CHO-K1 cells overexpressing SSTR4 (CHO-SSTR4) are purchased from DiscoveRx (product code 95-0059C2). The CHO-SSTR4 cells are maintained in F12K media with 10% Fetal Bovine Serum (Hyclone), 1% Pen/Strep (Life Technologies), and 800 μg/mL G418 (Life Technologies). To perform the assay, 3000 cells are plated per well in white 384-well plate (Corning 3570) in 50 μL complete media and the cells are allowed to attach for 16 hours in a 37° C., 5% CO2 incubator. The next day, the culture media is removed from the cells and the cells are washed (added then removed) with Kreb's Ringer Buffer (ZenBio, KRB-1000 mL). Test compounds are suspended in DMSO and diluted in stimulation buffer: Kreb's Ringer Buffer plus 0.5% BSA (Roche), 300 μM IBMX (Sigma), and 350 nM forskolin (Sigma). The cells are incubated in L compound/stimulation buffer for 30 minutes at room temperature. Cellular cAMP levels are detected with a HTRF LANCE Ultra cAMP kit (Perkin Elmer, catalog number TRF0264).The assay is performed in accordance with the manufacturer's instructions. Five μL of diluted Eu-W8044 labeled streptavidin (dilution: 1:50 in cAMP Detection Buffer) is added to each well. Then 5 μL of diluted biotin cAMP (dilution: 1:150 in cAMP Detection Buffer) is added to each well. The plates are covered and allowed to incubate for 60 minutes at room temperature on a shaker. HTRF (665 nm/615 nm) is read on a Perkin Elmer ENVISION plate reader. The pEC50 values are generated using Activity Base for Screening Data Management.
Measurement: EC50 = 5.01 nM (pChEMBL 8.300000190734863)
Descriptors: MW 411.55, ALogP 2.62, HBA 6, HBD 1, PSA 72.28, QED 0.76
Compound (SMILES): COCC(=O)N[C@@H]1CCC[C@H](C(=O)Nc2cc(-c3cc(F)c4nn(C)c(C(C)C)c4c3)c(C)cn2)C1
InChIKey: VUKIOSRRSWOLGW-PKOBYXMFSA-N
Target: CDK2/Cyclin A2 (PROTEIN COMPLEX), Homo sapiens
UniProt: P20248
Assay: CDK9/CyclinT1 Enzymatic Activity Assay: The inhibitory activity of compounds was evaluated in vitro using TR-FRET assay with white 384-well low volume microplate (Greiner Bio-One). CDK9/Cyclin T1 catalyzed phosphorylation of peptide in the presence and absence of compounds was measured and used in IC50 determination. Recombinant protein complex CDK9/Cyclin T1, expressed from insect cell, was purchased from ProQinase. Testing compounds were dissolved in DMSO at 1 mM and tested in 9-dose IC50 mode. The reaction mixture was prepared by mixing CDK9/CyclinT1 (1 nM final), ULight-4E-BP1 (50 nM final, Perkinelmer, TRF0128-D), and ATP (1 mM final) in assay buffer (20 mM of HEPES pH 7.4, 1 mM of EGTA, 0.05% BSA, 0.005% Tween 20, and 1 mM TCEP). The compound of interest in DMSO was added to each well in 3-fold serial dilution by dispenser (TECAN D300E) to make a 9.9 μL of reaction mixture. After 20 minutes preincubation at room temperature, 0.1 μL MgCl2(10 mM final) was added to initiate the reaction. Following a 45 minutes incubation at 37° C., the reaction was stopped by addition of 2 μL of quenching buffer consisting of Lance detection buffer (Perkinelmer CR97-100C), LANCE Ultra Europium-anti-P-4E-BP1 (Perkinelmer, TRF0216-D), EDTA, and incubate at room temperature for additional 60 minutes in dark. The reaction signal was measured by Envision multimode plate reader (PerkinElmer, 2102-0010). IC50values were determined by fitting the data to the standard 4 parameters with Hill Slope using GraphPad Prism software. See Table B (CDK9_T1).
Measurement: IC50 = 1310 nM (pChEMBL 5.880000114440918)
Descriptors: MW 495.6, ALogP 4.47, HBA 6, HBD 2, PSA 98.14, QED 0.5
Compound (SMILES): COc1c(NC(=O)Nc2ccc(Oc3ccnc(Nc4ccccc4)n3)c3ccccc23)cc(C(C)(C)C)cc1OCP(C)(C)=O
InChIKey: WRUIJSVPESNZTL-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase Src (SINGLE PROTEIN), Homo sapiens
UniProt: P12931
Assay: 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 μL) is incubated with the test compound (either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL, 2.5 μL each) for 2 hr at RT. The FRET peptides (8 μM, 2.5 μL), and appropriate ATP solutions (2.5 μL, 800 μM for c-Src, and 60 μM ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: IC50 = 61 nM (pChEMBL 7.210000038146973)
Descriptors: MW 639.69, ALogP 9.07, HBA 8, HBD 3, PSA 123.7, QED 0.13
Compound (SMILES): CCc1nc2ccc(C3CCN(CC(=O)N4CC(O)C4)CC3)nn2c1N(C)c1ccnc(-c2ccc(F)cc2)n1
InChIKey: GGYWYPCKXXWHIY-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Biochemical Assay: Compound IC50 values are determined in a fluorescent hENPP2 (UniProtKB/SwissProt Sequence ref Q13822) biochemical assay using the fluorogenic autotaxin substrate FS-3 as substrate. FS-3 is a doubly labeled analog of LPC wherein the fluorophore is quenched through intramolecular energy transfer. Without hENPP2, the emission of the probe is quenched. If the substrate is hydrolyzed by hENPP2, the emission of the probe is not quenched anymore resulting in a fluorescence increase. Inhibition of hENPP2 by compounds will result in a decrease of the signal. 10 uL of a dilution series of compound, starting from 20 uM highest concentration, dilution, is added to the wells. hENPP2 is used at a final concentration of 0.4 ug/mL or 0.64 ug/mL (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration). The enzyme is diluted in 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% fatty acid free BSA in a total volume of 20 uL. Enzyme mixture is added to compounds and the resulting mixture is incubated for 30 min at room temperature under shaking. The reaction is started by the addition of 20 uL of 0.75 uM FS-3 diluted in the same buffer as described above and the mixture is incubated at 30° C. for 30 min. Fluorescence is read on the Envision (Excitation 485 nm, emission 520 nM).
Measurement: IC50 = 5050 nM (pChEMBL 5.300000190734863)
Descriptors: MW 544.64, ALogP 3.04, HBA 9, HBD 1, PSA 102.99, QED 0.38
Compound (SMILES): C[C@H]1CC[C@H](Cn2c(C(F)c3ccccc3F)cc3nc(-c4noc(=O)[nH]4)nc(-c4cncc(Cl)c4)c32)CC1
InChIKey: MDDWTEBQIZRNHB-WSNUEFLKSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: Methods: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (SEQ ID NO:1: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards the p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat#Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 551, ALogP 6.51, HBA 7, HBD 1, PSA 102.49, QED 0.26