lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nc(N)ccc1CNC(=O)[C@H](C)NC(=O)[C@H]1C[C@@H](c2ccccc2)CCN1
InChIKey: LRFOMGAFAOKBNH-VDZJLULYSA-N
Target: Mannan-binding lectin serine protease 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00187
Assay: Enzymatic Assay for MASP-2: The assay was run at room temperature in an assay buffer containing 20 mM Hepes, pH 7.4, 140 mM NaCl and 0.1% Tween 20. Assay parameters were adjusted such that the assay was linear with respect to time, enzyme and substrate concentrations. Under these optimized assays conditions, IC50 values were equivalent to Ki values, except in a few cases of tight binding inhibitors. Cases of tight binding or possible slow binding inhibitors were handled by the methods described in Copeland R. A. (2013) Evaluation of Enzyme Inhibitors in Drug Discovery. 2nd Ed., John Wiley and Sons, Inc., Chapters 5-7.
Measurement: Ki < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 395.51, ALogP 1.63, HBA 5, HBD 4, PSA 109.14, QED 0.59
Compound (SMILES): C=CC(=O)N1CCC[C@H]1c1nc(-c2ccc(Oc3cccc(C)c3C)cc2)c2cnccn12
InChIKey: ADMCZFZVXVCJOO-QFIPXVFZSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Assay: Btk and Other Kinases: Btk kinase activity was determined using a homogenous time resolved fluorescence (HTRF) methodology. Measurements were performed in a reaction volume of 15 μL using 384-well assay plates. Kinase enzyme, inhibitor, ATP and 1 μM peptide substrate were incubated in a reaction buffer compose of Hepes50 mM (pH7.0), NaN3 0.02%, BSA 0.01%, Orthocanadate 0.1 mM. After one hour, the kinase reaction was quenched by the addition of Eμ-labeled antibody and XL-665 in 1×Detection buffer containing 60 mM EDTA (Cisbio), and the mixture was allowed to incubate for one hour. The HTRF signal was measured on a multimode plate reader (EnVision® Multilabel Reader, Perkin Elmer) with an excitation wavelength (2Ex) of 330 nm and detection wavelengths (λEm) of 615 and 665 nm. Activity was determined by the ratio of the fluorescence at 665 nm to that at 615 nm. For each compound, enzyme activity as measured at various concentrations of compound, Negative control reactions were performed in the absence of inhibitor in two replicates and eight no enzyme controls were used to determine baseline fluorescence levels.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 438.53, ALogP 5.65, HBA 5, HBD 0, PSA 59.73, QED 0.38
Compound (SMILES): N#Cc1c[nH]c2nccc(N3CCCC3NN)c12
InChIKey: BLMKBLDBMAAGCC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase MRCK beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5S2
Assay: Kinase Assay: MRCKα, MRCKβ, ROCK1 and ROCK2 assays were performed using an IMAP fluorescence polarization assay format (Molecular Devices Inc.). 8-12 nM of each kinase (Life Technologies) was incubated for 60 min at room temperature with 100 nM FAM-S6-ribosomal protein derived peptide (synthesized by Alta Biosciences, University of Birmingham UK) in the presence of 1 μM ATP and 0.5 mM MgCl2 in 20 mM Tris buffer (pH 7.4) containing 0.01% Tween-20 and 1 mM DTT (MRCKα and β); or 1 μM ATP, 10 mM MgCl2 in 20 mM Tris buffer (pH 7.5) containing 0.25 mM EGTA 0.01% Triton X-100 and 1 mM DTT (ROCK1 and ROCK2). Typically, dose response analyses were performed over concentration ranges from 0.005-100 μM. Reactions were stopped by adding 2 assay volumes of 0.25% (v/v) IMAP binding reagent in 1×IMAP binding buffer A (Molecular Devices). After 30 min incubation to allow binding reagent to bind phosphorylated peptide, fluorescence polarization was measured on a Tecan Saphire2 plate reader at excitation (470 nm) and emission (530 nm) wavelengths. Inhibition was calculated using no inhibitor and no enzyme controls as 0 and 100% inhibition, respectively.
Measurement: Ki = 29.7 nM (pChEMBL 7.53000020980835)
Descriptors: MW 242.29, ALogP 0.82, HBA 5, HBD 3, PSA 93.76, QED 0.54
Compound (SMILES): COc1ccc(Cn2cc(C(=O)Nc3cc(-c4[nH]c5c(c4Nc4ccccc4)C(=O)CC(C)(C)C5)ccn3)cn2)cc1
InChIKey: DNGYUAJEFVWZDC-UHFFFAOYSA-N
Target: Mitotic checkpoint serine/threonine-protein kinase BUB1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43683
Assay: Kinase Assay: In a typical assay 11 different concentrations of each compound (0.1 nM, 0.33 nM, 1.1 nM, 3.8 nM, 13 nM, 44 nM, 0.15 μM, 0.51 μM, 1.7 μM, 5.9 μM and 20 μM) were tested in duplicate within the same microtiter plate. To this end, 100-fold concentrated compound solutions (in DMSO) were previously prepared by serial dilution (1:3.4) of 2 mM stocks in a clear low volume 384-well source microtiter plate (Greiner Bio-One, Frickenhausen, Germany), from which 50 nl of compounds were transferred into a black low volume test microtiter plate from the same supplier. Subsequently, 2 μL of Bub1 (the final concentration of Bub1 was adjusted depending on the activity of the enzyme lot in order to be within the linear dynamic range of the assay: typically 200 ng/mL were used) in aqueous assay buffer [50 mM Tris/HCl pH 7.5, 10 mM magnesium chloride (MgCl2), 200 mM potassium chloride (KCl), 1.0 mM dithiothreitol (DTT), 0.1 mM sodium ortho-vanadate, 1% (v/v) glycerol, 0.01% (w/v) bovine serum albumine (BSA), 0.005% (v/v) Trition X-100 (Sigma), 1× Complete EDTA-free protease inhibitor mixture (Roche)] were added to the compounds in the test plate and the mixture was incubated for 15 min at 22° C. to allow pre-equilibration of the putative enzyme-inhibitor complexes before the start of the kinase reaction, which was initiated by the addition of 3 μL 1.67-fold concentrated solution (in assay buffer) of adenosine-tri-phosphate (ATP, 10 μM final concentration) and peptide substrate (1 μM final concentration). The resulting mixture (5 μL final volume) was incubated at 22° C. during 60 min., and the reaction was stopped by the addition of 5 μL of an aqueous EDTA-solution (50 mM EDTA, in 100 mM HEPES pH 7.5 and 0.2% (w/v) bovine serum albumin) which also contained the TR-FRET detection reagents (0.2 μM streptavidin-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-phosho-Serine antibody [Merck Millipore, cat. #35-002] and 0.4 nM LANCE EU-W1024 labeled anti-mouse IgG antibody [Perkin-Elmer, product no. AD0077, alternatively a Terbium-cryptate-labeled anti-mouse IgG antibody from Cisbio Bioassays can be used]). The stopped reaction mixture was further incubated 1 h at 22° C. in order to allow the formation of complexes between peptides and detection reagents.
Measurement: IC50 = 5.09 nM (pChEMBL 8.289999961853027)
Descriptors: MW 560.66, ALogP 6.48, HBA 7, HBD 3, PSA 113.93, QED 0.2
Compound (SMILES): CC(C)[C@H](NC(=O)Cc1cccc(Oc2ccccc2)c1)C(=O)N[C@@H](Cc1ccccc1)[C@@H](O)CCC(=O)N1CCc2ccccc21
InChIKey: XTHGKKUJTGJYJV-CGJJMRMRSA-N
Target: Renin (SINGLE PROTEIN), Homo sapiens
UniProt: P00797
Assay: n-Vitro Fluorescence Assay for Identification of Renin-Inhibitory Activity: In order to identify modulators of renin activity, a continuous enzymatic test was carried out with a synthetic peptide which carries a fluorescent group Edans (=(5-(aminoethyl)aminonaphthalenesulfonate) which is quenched by energy transfer from a Dabcyl (4′-dimethylaminoazobenzene-4-carboxylate) group on the same molecule, in Greiner 384-well microtitre plates. Cleavage of the peptidic substrate by renin causes an increase in the fluorescence intensity. In order to determine the efficacy of substances, the time-dependent increase in the fluorescence intensity in the presence of the substance was compared with the time-dependent increase in fluorescence in the absence of substances. The reference substance used was renin inhibitor 2 (Z-Arg-Arg-Pro-Phe-His-Sta-Ile-His N-Boc-Lys methyl ester Z) (Sigma-Aldrich). The substrate used was renin FRET substrate I (DABCYL-g-Abu-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr EDANS) (Anaspec, Fremont Calif.). The enzyme employed was recombinant human renin (Proteos, Kalamazoo, Mich.) in a final concentration of 10 nM. The test was carried out in 50 mM Mops buffer, 1.5% (v/v) of DMSO, 0.1% (w/v) of Igepal®, pH 7.2, 0.5% (w/v) of BSA. 2 μl of each substance solution with serially diluted substance concentration were added to in each case 4 μl of renin solution and incubated at room temperature for 15 min. The reaction was started by addition of 4 μl of substrate solution (final concentration 5 μM). After carrying out a starting-point fluorescence measurement (excitation wavelength 340 nm/emission wavelength 495 nm) using an Envision multilabel reader (Perkin Elmer), the reaction was incubated at 37° C. for 60 min. The amount of peptide fragment cleaved off during the reaction time was subsequently measured by determination of the increase in the fluorescence intensity at 495 nm (excitation wavelength 340 nm).
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 633.79, ALogP 5.62, HBA 5, HBD 3, PSA 107.97, QED 0.17
Compound (SMILES): CCn1c(-c2c(Cl)cccc2Cl)nn(-c2cc(OC)c(C(=O)Nc3cccc(C(F)(F)F)c3)cc2F)c1=O
InChIKey: ZIUJOSBFVKVFIT-UHFFFAOYSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Enzymatic Assay1: The enzymatic assay couples DHODH activity with bleaching of the dye 2,6-dichlorophenolindophenol (DCIP) (Knecht and Loffler, 1998; Miller et al., 1968). The assay was conducted in buffer containing 50 mM Tris, 0.1% Triton X-100, 150 mM potassium chloride, 2 nM DHODH, 1 mM dihydroorotate, 0.1 mM decylubiquinone, 0.06 mM DCIP, and 2% DMSO at pH 8.0 at 32 degree celsius. The reaction was initiated by addition of substrates. Enzyme activity was monitored kinetically by the reduction in DCIP absorbance at 600 nm. Purified recombinant human DHODH enzyme was purchased from Novus (cat. no. NBP1-98916). Other chemicals were purchased from Sigma-Aldrich. Absorbance measurements were obtained using a BMG clarion star plate-reading spectrophotometer.
Measurement: IC50 = 30000 nM (pChEMBL 4.519999980926514)
Descriptors: MW 569.34, ALogP 6.45, HBA 6, HBD 1, PSA 78.15, QED 0.27
Compound (SMILES): C[C@@H]1C[C@H](NC(=O)c2cc(C3CC3)on2)CCN1S(=O)(=O)CC1CCN(CCN2CCOCC2)CC1
InChIKey: OKWIJOVNWPCIIC-DENIHFKCSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).% Inhibition Calculation% ⁢ ⁢ inh = 100 - ( dpm cmpd - dpm min dpm max - dpm min ) × 100Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.Four-Parameter IC50 FitY = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ CoefficientWhere top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
Measurement: IC50 = 111 nM (pChEMBL 6.960000038146973)
Descriptors: MW 523.7, ALogP 1.51, HBA 8, HBD 1, PSA 108.22, QED 0.52
Compound (SMILES): COCCN1CCN2C(=O)N(c3c(F)cc(C#Cc4ccccc4)cc3F)C(=O)C[C@@]2(C)C1=O
InChIKey: ONDDJVPUWJOWBR-VWLOTQADSA-N
Target: Metabotropic glutamate receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14833
Assay: Ca2+ Mobilization In Vitro Assay: A monoclonal HEK-293 cell line stably transfected with a cDNA encoding for the human mGlu4 receptor was generated; for the work with mGlu4 Positive Allosteric Modulators (PAMs), a cell line with low receptor expression levels and low constitutive receptor activity was selected to allow the differentiation of agonistic versus PAM activity. Cells were cultured according to standard protocols (Freshney, 2000) in Dulbecco's Modified Eagle Medium with high glucose supplemented with 1 mM glutamine, 10% (vol/vol) heat-inactivated bovine calf serum, Penicillin/Streptomycin, 50 μg/ml hygromycin and 15 μg/ml blasticidin (all cell culture reagents and antibiotics from Invitrogen, Basel, Switzerland).About 24 hrs before an experiment, 5×104 cells/well were seeded in poly-D-lysine coated, black/clear-bottomed 96-well plates. The cells were loaded with 2.5 μM Fluo-4AM in loading buffer (1×HBSS, 20 mM HEPES) for 1 hr at 37° C. and washed five times with loading buffer. The cells were transferred into a Functional Drug Screening System 7000 (Hamamatsu, Paris, France), and 11 half logarithmic serial dilutions of test compound at 37° C. were added and the cells were incubated for 10-30 min. with on-line recording of fluorescence. Following this pre-incubation step, the agonist (2S)-2-amino-4-phosphonobutanoic acid (L-AP4) was added to the cells at a concentration corresponding to EC20 with on-line recording of fluorescence; in order to account for day-to-day variations in the responsiveness of cells, the EC20 of L-AP4 was determined immediately ahead of each experiment by recording of a full dose-response curve of L-AP4.
Measurement: EC50 = 137 nM (pChEMBL 6.860000133514404)
Descriptors: MW 467.47, ALogP 2.77, HBA 4, HBD 0, PSA 70.16, QED 0.65
Compound (SMILES): COc1cc2nccc(Oc3ccc(NC(=O)c4cn(CCN5CCCCC5)c(=O)n(-c5ccc(F)cc5)c4=O)cc3F)c2cc1OC
InChIKey: NUNGWJQKZDOGAA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Kinase Assay: The ability of compounds to inhibit the kinase activity of recombinant human baculovirus-expressed AXL was measured by homogeneous TRF (HTRF) using Cisbio's KinEASE assay system in white 384-well Optiplates. Assay buffer contained 1 mM DTT, 2 mM MnCl2, 2% DMSO, 50 nM supplement enzymatic buffer, and 1× enzymatic buffer. A 2× concentration of tyrosine kinase (TK) substrate-biotin/ATP mixture made in assay buffer was added to plates at 10 μL/well using the Multidrop Combi (Thermo Fisher Scientific, Waltham, Mass.). The final concentrations were 0.3 μM TK substrate-biotin, and 1.3 μM ATP. Compounds (100 nL), diluted in 100% DMSO on the Biomek FX, (Beckman Coulter, Inc., Brea, Calif.), were transferred to the assay plates using the Biomek FX pintool (2.5% final DMSO in assay). A 2× concentration (final=12 ng/mL) of GST-AXL (diluted in assay buffer) was added to plates at 10 uL/well using the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 30 minutes. A 4× stock of Streptavidin-XL665 (final=18.8 nM) and a 1:100 diluted stock of TK antibody-cryptate were made in HTRF detection buffer and mixed together just prior to adding 20 μL/well on the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 1 hour. The fluorescence of the resulting solution was measured using the PerkinElmer EnVision 2102 multi-label plate reader (PerkinElmer, Waltham, Mass.) with an excitation wavelength of 337 nm (laser) and emission wavelengths of 590 and 665 nm. Raw data was expressed as the ratio of 665/590×10,000.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 657.67, ALogP 5.37, HBA 10, HBD 1, PSA 116.92, QED 0.21
Compound (SMILES): CN1C(=O)C(NC(=O)c2cc(Oc3ccccc3)ccn2)COc2ccc(C#CC3(O)CCCC3)cc21
InChIKey: QHIKGWRYFLYBHL-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: biochemical assay using the ADP-Glo technology: ADP-Glo (Promega, Madison, Wis., USA) reagents were thawed at ambient temperature. Kinase Detection Reagent was prepared by mixing kinase detection buffer with the lyophilized kinase detection substrate.A 500 ml stock volume of 5× Reaction Kinase Buffer was made by mixing 1000 μl of 1M MgCl2, 500 μl of 1M Tris-HCL pH7.4, 0.5 mg/ml (25 mg) of BSA, and 3475 μl of distilled H2O. A 3 ml 2× working stock volume of Reaction Kinase Buffer was made containing a final concentration of 100 μM DTT and 4 mM MnCl2.Components of RIPK1 enzyme (Rigel Pharmaceuticals, South San Francisco, Calif., USA) were thawed on ice. Diluted RIPK1 was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer) to 31 ng/well. A 166 μM working stock ATP assay solution was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer).Compounds were serially diluted in DMSO from 250 uM in 4-fold dilutions then diluted 1:5 in 2× Reaction Buffer in a 96 well plate. 1.0 μl of diluted compound was added to a 384 well plate in duplicate. 2 μl of diluted Active RIPK1 was added to 384 well plate (do not add to column 1) add 2× rxn buffer to column 1. AKT (Anaspec, Fremont, Calif., USA) at 150 nM was combined with ATP working stock at equal volume and 2 ul/well were added to the 384 well plate. The final reaction volume was 5.0ul.The plate was quickly centrifuged and the reaction was incubated at 30° C. for 30 minutes. Adding 5 μl of ADP-Glo terminated the reaction. The plate was quickly centrifuged and the reaction was incubated at room temperature for 40 minutes. Kinase Detection Reagent was then added and incubated at room temperature for 30 minutes. The relative light unit (RLU) of kinase reaction was determined by luminescent (Luminescence 0.1 s) using a Wallac Victor2 Luminometer (PerkinElmer, Waltham, Mass., USA).
Measurement: IC50 = 29.6 nM (pChEMBL 7.53000020980835)
Descriptors: MW 497.55, ALogP 3.68, HBA 6, HBD 2, PSA 100.99, QED 0.53
Compound (SMILES): Cc1ccc(NC(=O)N2CC[C@@H](CC(F)(F)F)C2)cc1-c1cc(N2CCOCC2)nc(N2C[C@H](O)CC2=O)c1
InChIKey: QAPCEEGFBAEKNS-GHTZIAJQSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Enzymatic RAF1 Kinase: Small molecule inhibition of RAF1 kinases was measured using ADP-Glo assay. In the assay, ADP is converted to ATP in the presence of test kinase and substrate, resulting in luciferase reaction and luminescent readout with light generated proportional to the relative kinase activity. Compounds diluted in DMSO were used in 10-point, 3-fold dose curve for both assays. Final concentrations of 3 nM RAF1 (CarnaBio, Cat. 09-125) and 30 nM MEK1 substrate (Millipore, Cat. 14-420) were incubated with 3 μM ATP, 10 mM MgCl2, 0.003% Brij-35, 2 mM DTT, 0.05% BSA, 1 mM EGTA, and 50 mM HEPES for 90 minutes at room temp prior to addition of ADP-Glo reagent (Promega, Cat. V9102) for 40 minutes, and detection reagent for 45 minutes. Luminescence was read on an Envision plate reader (PerkinElmer) and percent remaining activity was used to calculate IC50 using a four-parameter fit model using Dotmatics Knowledge Solutions Studies curve fitting (Dotmatics, Bishops Stortford, UK, CM23).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 547.58, ALogP 3.8, HBA 6, HBD 2, PSA 98.24, QED 0.59
Compound (SMILES): N#CC[C@@H](C1CC1)n1nc(Nc2ccc3c(c2)OCO3)c2c(=O)[nH]ccc21
InChIKey: LZENBEADSYVOQL-ZDUSSCGKSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Jak Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format 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 μL of 1.11× enzyme and 1.11× substrate in 1× 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 μL of 10×ATP in 1× 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 μL of 2× 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. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 990 nM (pChEMBL 6)
Descriptors: MW 363.38, ALogP 3.06, HBA 7, HBD 2, PSA 104.96, QED 0.72
Compound (SMILES): COc1cc2[nH]c(=O)c([C@H](C)Nc3ccc(C#N)c(C)n3)cc2cc1Cl
InChIKey: MJIIMSGVGMGBRO-NSHDSACASA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: IDH1-R132H and IDH1-R132C Enzymatic Assay: Assays were performed in a 384-well black plate. An aliquot of 250 nL of compound was incubated with 10 μL of 30 nM IDH1-R132H or 10 nM IDH1-R132C recombinant protein in assay buffer (50 mM Tris pH=7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% (w/v) Bovine Serum Albumin, and 0.01% Triton X-100) in each well at 25° C. for 15 minutes. After the plate was centrifuged briefly, an aliquot of 10 μL of 2 mM α-ketoglutarate and 20 μM NADPH solution prepared in assay buffer was then added to each well and the reaction was maintained at 25° C. for 45 minutes. An aliquot of 10 μL of diaphorase solution (0.15 U/mL diaphorase and 30 μM Resazurin in assay buffer) was added to each well. The plate was maintained at 25° C. for 15 minutes and then read on a plate reader with excitation and emission wavelengths at 535 nm and 590 nm, respectively. The IC50 of a given compound was calculated by fitting the dose response curve of inhibition of NADPH consumption at a given concentration with the four parameter logistic equation.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 368.82, ALogP 3.94, HBA 5, HBD 2, PSA 90.8, QED 0.73
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(C)c(Br)nn2-c2ccccc2)[C@H](c2ccc(F)cc2)C1
InChIKey: TVUOMYQLSBGGLU-LEWJYISDSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA Omnia Assay: Trk enzymatic selectivity was assessed using Omnia Kinase Assay reagents from Invitrogen Corp. Enzyme (TrkA from Invitrogen Corp.) and test compound (various concentrations) were incubated for 10 minutes at ambient temperature in a 384-well white polypropylene plate (Nunc catalog#267462). Omnia Tyr Peptide #4, as well as ATP, were then added to the plate. Final concentrations were as follows: 20 nM enzyme, 500 μM of ATP, 10 μM peptide substrate. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The production of phosphorylated peptide was monitored continuously for 70 minutes using a Molecular Devices FlexStation II384 microplate reader (excitation=360 nm; emission=485 nm). Initial rates were calculated from the progress curves. IC50 values were calculated from these rates using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 516.42, ALogP 4.32, HBA 5, HBD 2, PSA 71.42, QED 0.49
Compound (SMILES): CCC(=O)NC1CCN(c2nc3ccc(-c4c(C)noc4C)c4c3n2[C@@H](c2ccccn2)CO4)CC1
InChIKey: KJSYGXDCWXTSEZ-JOCHJYFZSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 20 μL for BD1 and 40 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature for 75 min. in the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% CHAPS, 0.01% BSA), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4), 3.8 nM (BRD4-BD1, BPS Bioscience #31040) or 20 nM (BRD4-BD2, BPS Bioscience #31041). The reaction followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at 4 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 486.58, ALogP 4.18, HBA 8, HBD 1, PSA 98.31, QED 0.45
Compound (SMILES): Cc1cccc(F)c1-c1cc2c(NC(=O)c3ccc(N4CCN(C)CC4)cc3)n[nH]c2cn1
InChIKey: PDFFOJZXXFYTMX-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 μL of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 μl of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 μl of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 444.51, ALogP 4.08, HBA 5, HBD 2, PSA 77.15, QED 0.5
Compound (SMILES): O=C(NCC1CCC(F)(F)CC1)c1cn(CC(=O)N2CCOCC2)c2cccc(Cl)c12
InChIKey: MLGGONKYUOQNCS-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Pore Permeation Assay: Agonist-induced pore formation was determined by measuring cellular uptake of YO PRO fluorescence dye in HEK293 transfected with human P2X7 receptor. A HEK293 cell over expressing human P2X7 was harvested using HQTase reagent to detach the cells from T75 cm flask. The harvested cells are centrifuged @1200 rpm for 5 min at room temperature. The viability of cells was determined by Trypan blue dye and the cells are plated @10,000 cell/well in 50 ul volume in a 384W BD Poly lysine coated plate and incubated overnight at 37 C. After overnight incubation, the culture medium was replaced with 35 ul/well assay buffer (5 mM KCl, 0.1 mM CaCl2, 5 mM Glucose, 10 mM HEPES buffer pH7.4 containing 125 mM NaCl. The serial dilution of compounds was performed using Bravo liquid handling instrument and the compounds were added using Bravo to the cell assay plate starting at 2.5 uM with three dilutions for 10 points. The positive control inhibitor compound was added to column 23. The plate was shaken slowly on a plate shaker for 10 seconds. The cells were incubated with the compound for 20 minutes at room temperature. After the incubation period, YO PRO dye (1 uM) along with BzATP (10 uM) were added to cells at 10 ul/well. The plate was centrifuged at 1000 rpm for 5 seconds and incubated at room temperature for 30 minutes. The uptake of YO PRO dye into the cells was measured using Envision Fluorescence plate reader instrument (Perkin Elmer).
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 453.92, ALogP 3.71, HBA 4, HBD 1, PSA 63.57, QED 0.75
Compound (SMILES): Cc1cc(OCC2CCC2)cnc1-c1cc(F)cc(OCc2ccc(C(=O)NS(C)(=O)=O)cc2)c1
InChIKey: KKHFCIRTSASKAA-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: EFS-FRET Assay: This screen is used to determine the effects of compounds on human Nav1.7 channels, utilizing electrical field stimulation (EFS) system in 96-well plate format on FDSS (Hamamatsu Photonics) platform. The changes of membrane potential are monitored with FRET dye pair, DiSBAC2(3) and PTS 18.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 498.58, ALogP 4.64, HBA 6, HBD 1, PSA 94.59, QED 0.46
Compound (SMILES): COc1cc(C(=O)N2[C@@H](c3ccccc3Cl)CC[C@H]2C(=O)O)ccc1N1CCCC1
InChIKey: GKOGZNPFRUPJOW-QUCCMNQESA-N
Target: Free fatty acid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O15552
Assay: BINDING ASSAY: The following assay can be used for determination of GPR43 activation. When a GPCR is in its active state, either as a result of ligand binding or constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and subsequent binding of GTP to the G protein. The alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyses the GTP to GDP, at which point the receptor normally is deactivated. Activated receptors continue to exchange GDP for GTP. The non-hydrolysable GTP analog, [35S]GTPγS, was used to demonstrate enhance binding of [35S]GTPγS to membranes expressing receptors. The assay uses the ability of GPCR to stimulate [35S]GTPγS binding to membranes expressing the relevant receptors. The assay can, therefore, be used in the direct identification method to screen candidate compounds to endogenous or not endogenous GPCR.
Measurement: EC50 = 600 nM (pChEMBL 6.21999979019165)
Descriptors: MW 428.92, ALogP 4.38, HBA 4, HBD 1, PSA 70.08, QED 0.76
Compound (SMILES): CNC1CC=C(c2cnc(N)c3c(-c4ccc(NS(=O)(=O)c5cccc(F)c5)c5ccccc45)nc(C(C)C)n23)CC1
InChIKey: FRIUZDNCRNYJLA-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase/endoribonuclease IRE1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75460
Assay: Biochemical Assay: The kinase reactions were performed in 384 well white ProxiPlate-384 Plus plates (PERKIN Elmer 6008280) using 25 mM MOPS assay buffer with 1 mM dithiothreitol, 25 mM MgCl2, 12.5 mM i-glycerophosphate, 5 mM EGTA, and 50 ug/mL BSA. Test compounds were prepared on the day of assay and dispensed using D300 digital dispenser as a 10-point 1/2 log dilution series in duplicate, normalised to a final DMSO concentration of 3%. Test compounds were pre-incubated for 30 min at room temperature with 10 nM IRE1alpha kinase (E31-11G from Signal Chem) in 2.5 uL of assay buffer and the reaction started by addition of 2.5 uL of ATP in assay buffer, to give a final ATP concentration of 100 uM and 5 nM IRE1alpha kinase. After 4 hours incubation at room temperature the reactions were stopped and the kinase activity determined using the ADP-Glo reagent from Promega, according to the manufacturer's instructions. Luminescence was measured on a luminometer (EnVision, PerkinElmer) and IC50 values calculated by fitting a sigmoidal curve to percent inhibition of control versus Log 10 of compound concentration.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 584.72, ALogP 6.35, HBA 7, HBD 3, PSA 114.41, QED 0.21
Compound (SMILES): C[C@H](N)c1cccc(-c2cc(-c3ncc(C(F)(F)F)s3)c3cnn(-c4cccc(CO)n4)c3c2)n1
InChIKey: QLMFGHTXOALITR-ZDUSSCGKSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biochemical Assay: The biochemical assay is in a AlphaScreen format. The kinase reaction is based on the IRAK-4 phosphorylation of a biotin labeled peptide. The phosphopeptide is incubated with anti-phosphothreonine antibody as well as streptavidin- and protein A-coated beads. Binding of the protein-A coated beads to the antibody and the streptavidin beads to the peptide, leads to an energy transfer from one bead to the other, ultimately producing a luminescent/fluorescent signal.Generally, the kinase reaction is carried out at 1 nM IRAK4, 1.6 μM peptide, 1 mM ATP in reaction buffer 50 mM Hepes, 60 mM NaCl, 5 mM MgCl2, 0.25 mM MnCl2, 2 mM DTT, 0.01% BSA, 0.01% Tween-20) for 3.5 h at RT.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 496.52, ALogP 5.14, HBA 8, HBD 2, PSA 102.74, QED 0.35
Compound (SMILES): Cn1cc(-c2cnn(Cc3ccccc3)c2)c(OCCC(F)(F)F)cc1=O
InChIKey: CMMUUSFMDAEUMA-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: In Vitro CBP Inhibition: The CBP-inhibitory activity of the compounds described herein was determined by calculating the IC50. More specifically, CBP inhibitor activity was assayed as follows: CBP was cloned and expressed in E. coli as His-tag protein and purified by Nickel affinity and gel-filtration chromatography. The protein was further characterized as a single band with the correct molecular weight by SDS-PAGE. CBP binding and inhibition is assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Perkin Elmer). In a 384-well ProxiPlate CBP (50 nM final) was combined with peptide (20 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 min incubation at room temp, Alpha-streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 calculated using a four parameter non-linear curve fit.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 377.37, ALogP 3.63, HBA 5, HBD 0, PSA 49.05, QED 0.66
Compound (SMILES): C=CC(=O)Nc1cc(OCc2nccs2)cc(-c2ccc(C(N)=O)c3[nH]ccc23)c1
InChIKey: QXWYLVGNCUOEBV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) : The in-house BTK corresponds to recombinant human catalytic domain (aa 393-659), which was expressed in SF9 cells with an N-terminal his tag and purified by immobilized metal affinity chromatography. BTK was mixed with peptide substrate (biotin-TYR1, Sequence: Biotin-(Ahx)-GAEEEIYAAFFA-COOH, 0.2 μM final) at varying inhibitor concentrations in reaction buffer: 50 mM MOPSO pH 6.5, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTT, 0.01% BSA, 0.1 mM Na3VO4 and 0.001 mM ATP. After about 60 mM incubation at room temperature, the reaction was quenched by addition of EDTA (final concentration: 100 mM) and developed by addition of detection reagents (final approximate concentrations: 30 mM HEPES pH 7.0, 0.06% BSA, 0.006% Tween-20, 0.24 M KF, 80 ng/mL PT66K (europium labeled anti-phosphotyrosine antibody cat #61T66KLB Cisbio, Bedford, Mass.) and 0.6 μg/mL SAXL (Phycolink streptavidin-allophycocyanin acceptor, cat #PJ25S, Prozyme, San Leandro, Calif.). The developed reaction was incubated in the dark for about 60 mM at room temperature, then read via a time-resolved fluorescence detector (Rubystar, BMG) using a 337 nm laser for excitation and monitoring emission wavelength at 665 nm. Within the linear range of the assay, the observed signal at 665 nm was directly related to phosphorylated product and can be used to calculate the IC50 values.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 418.48, ALogP 4.09, HBA 5, HBD 3, PSA 110.1, QED 0.39
Compound (SMILES): Fc1ccc([C@H]2CC[C@H](CCNC3CCOCC3F)CC2)c(Cl)c1
InChIKey: VONXTIRDLATQHN-PHJIHURGSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Cell-Based NOP Assay: Formula (I) compounds were tested for antagonism of the nociceptin receptor (NOP) in a cell-based cAMP assay, using the LANCE cAMP Detection Kit, Perkin Elmer (Waltham, Mass.). This assay (LANCE cAMP) is a homogeneous, time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay designed to measure cAMP produced upon modulation of adenylyl cyclase activity by G protein-coupled receptors (GPCRs).ProtocolCells expressing human opioid NOP (ValiScreen cell line, cat #ES-230-C Perkin Elmer (Waltham, Mass.) were dispensed in simulation buffer (5 mM Hepes, 0.1% BSA, 0.5 mM IBMX in Hanks Buffered Saline Solution) at 2.5 μL cells per well (400 cells/well) into a 1536 white TC-treated assay plate. Compound was then added and plates were incubated for 15 minutes at room temperature. To each well was added 2.5 μL of a solution containing forskolin (3 μM final) and nociceptin peptide (390 pM final) diluted in stimulation buffer, and the plates were incubated for 30 minutes at room temperature. To each well was dispensed 5 μL of LANCE Ultra detection (1:300 dilution of anti-cAMP ULight with a 1:100 dilution of Eu-cAMP tracer diluted in cAMP Detection Buffer) reagent, and the plates were incubated for 1 hr at room temperature. The plate was then read on the ViewLux microplate reader (Perkin Elmer, Waltham, Mass.) using the LANCE 1536 protocol with 60 second exposure times and 50 μs delay. This protocol has an excitation wavelength of 340 nm (DUG11 filter) and emission wavelengths of 615 nm (Eu-Donor) and 665 nm (Alexa 647 cAMP antibody acceptor).Data AnalysisData was analyzed, normalized, and visualized using ACAS (John McNeil & Co.). Efficacy was calculated using activity of the vehicle (DMSO) as 0% efficacy and the activity of the corresponding reference positive control (SCH 221510, Tocris Bioscience (Pittsburgh, Pa.), Catalog number 3240) as 100% efficacy. Activity was calculated as the ratio of donor and acceptor emissions. All curve fits and IC50 determinations were performed using a non-linear regression model. The IC50 value was measured as the concentration at which half-maximal response was calculated.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 357.87, ALogP 4.86, HBA 2, HBD 1, PSA 21.26, QED 0.81
Compound (SMILES): Cn1ccc(C2CCN(S(=O)(=O)C[C@H]3C[C@@H](N(C)c4ncnc5[nH]ccc45)C3)CC2)n1
InChIKey: LNTSTFPYHHHPLZ-WOVMCDHWSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 0.066 nM (pChEMBL 10.180000305175781)
Descriptors: MW 443.58, ALogP 2.12, HBA 7, HBD 1, PSA 100.01, QED 0.63
Compound (SMILES): Nc1cnc(-c2ccc(-c3ccccc3N3CCCC3=O)cc2F)cn1
InChIKey: LMZPOUUBSVKRLS-UHFFFAOYSA-N
Target: Arachidonate 5-lipoxygenase-activating protein (SINGLE PROTEIN), Homo sapiens
UniProt: P20292
Assay: Binding Assay: The assay below is used to test the modulatory activity of compounds against FLAP. Human and mouse FLAP-encoding DNA was amplified by polymerase chain reaction and cloned into pFastBac1 (Invitrogen) with a NH2-terminal 6-His tag for expression in Spodoptera frugiperda (Sf-9) cells. FLAP-containing membranes were prepared as was a FITC-labeled FLAP modulator [5-[({[2-(2-{3-[3-(tert-Butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid]. The FLAP binding assay is performed in HTRF format (homogeneous time resolved fluorescence). FLAP-containing membranes (1 μg/well final for human) are incubated in the presence of the HTRF ligand, [5-[({[2-(2-{3-[3-(tert-butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid] (25 nM final), a terbium labeled anti-His tag antibody (0.5 ng/well final, from Cisbio) and compounds. The reaction is allowed to proceed for two hours after which the plate is read on an Envision plate reader in HTRF mode. Data are expressed as a HTRF ratio.
Measurement: Ki = 1479 nM (pChEMBL 5.829999923706055)
Descriptors: MW 348.38, ALogP 3.66, HBA 4, HBD 1, PSA 72.11, QED 0.78
Compound (SMILES): CCn1cc(C(=O)NCc2cccc(F)c2F)c2cc(OC)c(OC)cc2c1=O
InChIKey: MCLBTMAPJKKNDT-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Enzymatic Assay: The PDE enzymatic reaction was carried out in assay buffer (20 mM Tris-HCl pH7.5, 10 mM MgCl2, 0.1% bovine serum albumin) containing enzyme and substrate. The PDE enzymes concentration ranged from 10 pM 250 pM, depending on each enzyme's specific activity. The substrate cyclic nucleotide (cAMP or cGMP) concentration used in the assay was 20 nM for PDE10, and 100 nM for other PDEs. The inhibitory effect of compound was determined by incubating various concentration of inhibitor in the enzymatic assay. Typically, compound was serial diluted in DMSO then further diluted in assay buffer. Next, the compound at varying concentration was mixed with PDE enzyme. The reaction was initiated by addition of cyclic nucleotide substrate, and incubated for 60 minutes at 29 C. The reaction was stopped by addition of lysis buffer from assay kit. The cAMP-d2 and anti-cAMP cryptate in the lysis buffer detected the level of cAMP left from the PDE hydrolysis reaction. The PDE activity is reversely correlated with the amount of cAMP left in the reaction and can be converted to the percent activity of an uninhibited control (100%).
Measurement: IC50 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 402.4, ALogP 3.25, HBA 5, HBD 1, PSA 69.56, QED 0.69
Compound (SMILES): COCCOc1cc(-c2cccc(C3(N)CCC3)n2)cc2c1cnn2-c1cccc(CO)n1
InChIKey: NJPMSNVXYUGDRH-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biochemical Assay: The biochemical assay is in a AlphaScreen format. The kinase reaction is based on the IRAK-4 phosphorylation of a biotin labeled peptide. The phosphopeptide is incubated with anti-phosphothreonine antibody as well as streptavidin- and protein A-coated beads. Binding of the protein-A coated beads to the antibody and the streptavidin beads to the peptide, leads to an energy transfer from one bead to the other, ultimately producing a luminescent/fluorescent signal.Generally, the kinase reaction is carried out at 1 nM IRAK4, 1.6 μM peptide, 1 mM ATP in reaction buffer 50 mM Hepes, 60 mM NaCl, 5 mM MgCl2, 0.25 mM MnCl2, 2 mM DTT, 0.01% BSA, 0.01% Tween-20) for 3.5 h at RT.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 445.52, ALogP 3.34, HBA 8, HBD 2, PSA 108.31, QED 0.4
Compound (SMILES): CCOc1c(C(C)n2nc(C)c3c(N)ncnc32)cc(Cl)c(C)c1-c1ccc(C(=O)NC)nc1
InChIKey: SPDKDKKGRGWOLC-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Scintillation Proximity Assay: [γ-33P]ATP (10 mCi/mL) was purchased from PerkinElmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kδ (p110δ/p85α) was purchased from Millipore (Bedford, Mass.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from SigmaAldrich (St. Louis, Mo.). Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from GE healthcare life sciences (Piscataway, N.J.).The kinase reaction was conducted in polystyrene 384-well matrix white plate from Thermo Fisher Scientific in a final volume of 25 μL Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 0.5%. The PI3K assays were carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 20 μM ATP, 0.2 μCi [γ-33P] ATP, 4 nM PI31(6. Reactions were incubated for 210 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 150 mM potassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400 μM ATP. The final concentration of SPA beads was 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1800 rpm for 10 minutes, the radioactivity of the product was determined by scintillation counting on Topcount (PerkinElmer).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 479.97, ALogP 4.11, HBA 8, HBD 2, PSA 120.84, QED 0.43
Compound (SMILES): Cc1cc(C(F)(F)F)cc2c1nc(Cc1c(Cl)ccc(C(=O)N3C[C@@H]4[C@H](C3)[C@H]4C(=O)O)c1Cl)n2C
InChIKey: UWIISQJFSKTEHJ-QWHZDKJVSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: transient transactivation assay: The primary screen was performed by transient transactivation assays. These cell-based assays were carried out using Cos-7 cells transfected with a chimeric human RORγ-Gal4 receptor expression plasmid and a 5Gal4 pGL3 TK Luc reporter plasmid. Transfections were performed by a chemical agent (Jet PEI). Transfected cells were distributed in 384-wells plates and were allowed to recover for 24 h. The culture medium was then removed and fresh medium containing the compounds to be tested was added (final concentration ranging from 10−4 M to 3 10−10 M). After an overnight incubation, luciferase expression was measured by adding SteadyGlo according to the manufacturer's instructions (Promega). T0901317 at 10−5 M was used as reference. Results were expressed as fold induction compared to basal level or as percentage activity compared to references taken as 100% (basal) or 0% (T0901317). Dose-effect curves and IC50 were calculated using the software Assay Explorer (MDL).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 526.34, ALogP 5.2, HBA 4, HBD 1, PSA 75.43, QED 0.51