lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@@H]1COCCN1c1cc(-c2cnc3n2CCC3)c2ccnc(-c3ccnn3C3CCCCO3)c2n1
InChIKey: WYNLHHDAYYWSPU-YDONVPIESA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Time-Resolved Fluorescence Energy Transfer (TR-FRET Tracer A): To determine of binding activity of the test compounds, full-length human ATR protein was expressed and purified together with ATRIP as described above. Furthermore, a fluorescently labelled compound (tracer A) was used as a tracer molecule. Detection of the binding event of the tracer was achieved by time-resolved fluorescence energy transfer (TR-FRET). We used an anti-GST-Terbium antibody (CisBio) that binds to the GST-tag at the N-terminus of ATR-kinase. Excitation of Terbium with 337 nm light results in emission of fluorescent light with 545 nm. In case a tetrameric complex has formed (antiGST-Tb+GST-ATR+Strp2-ATRIP+tracer), part of the energy will be transferred from the Terbium to the fluorophore that itself emits light of 570 nm. Displacement of the fluorescent tracer by a test compound leads to a reduction of the TR-FRET-signal.For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384well microtiter plate (MTP, Greiner Bio-One, Frickenhausen, Germany). To prepare the ATR-working solution, ATR/ATRIP stock solution was diluted in assay buffer [50 mM HEPES (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% (w/v) Igepal, 0.01% (w/v) BSA] to 4.2 nM protein concentration (concentration may vary from lot to lot of protein preparation). AntiGST-Tb antibody was diluted to 4.2 nM. The ATR-working solution was incubated for 30 min at 22° C. prior to dispensing to pre-form the complex of antiGST-Tb+GST-ATR+ATRIP. Then, 3 μl of the ATR-working solution were added to the test compound and the mixture was incubated for 10 min at 22° C. to allow pre-binding of the test compounds to ATR/ATRIP. Then, 2 μl of a 100 nM solution of either tracer A or B in assay buffer were added to the ATR-working solution. The resulting mixture was incubated for 30 min at 22° C. The measurement of the TR-FRET signal was performed in a standard HTRF-compatible MTP reader instrument (e.g. BMG Pherastar) by recording the fluorescence emissions at 545 nm and 570 nm after excitation at 337-350 nm. The ratio between emission at 570 nm divided by emission at 545 nm was calculated to give the well ratio. The experimental data (well ratios) were normalised by the following way: positive control contained ATR-working solution plus either tracer A or B solution (=0% inhibition), the negative control contained all components except GST-ATR/ATRIP (=100% inhibition).
Measurement: IC50 = 10.1 nM (pChEMBL 8)
Descriptors: MW 485.59, ALogP 4.23, HBA 9, HBD 0, PSA 83.12, QED 0.43
Compound (SMILES): COC(=O)N(C)c1cc(-c2c(-c3ccc(C#N)cc3)nc(OC[C@@H]3CCCN(C)C3)c3nccn23)ccc1C
InChIKey: VHOINLKPERVOLE-JOCHJYFZSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LANCE LSD1/KDM1A Demethylase Assay: 10 μL of 1 nM LSD-1 enzyme (ENZO BML-SE544-0050) in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 25 mM NaCl, 5 mM DTT) were preincubated for 1 hour at 25° C. with 0.8 μL compound/DMSO dotted in black 384 well polystyrene plates. Reactions were started by addition of 10 μL of assay buffer containing 0.4 M Biotin-labeled Histone H3 peptide substrate: ART-K(Me1)-QTARKSTGGKAPRKQLA-GGK(Biotin) SEQ ID NO:1 (AnaSpec 64355) and incubated for 1 hour at 25° C. Reactions were stopped by addition of 10 μL 1× LANCE Detection Buffer (PerkinElmer CR97-100) supplemented with 1.5 nM Eu-anti-unmodified H3K4 Antibody (PerkinElmer TRF0404), and 225 nM LANCE Ultra Streptavidin (PerkinElmer TRF102) along with 0.9 mM Tranylcypromine-HCl (Millipore 616431). After stopping the reactions plates were incubated for 30 minutes and read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 524.63, ALogP 5.17, HBA 8, HBD 0, PSA 95.99, QED 0.34
Compound (SMILES): CC(C)(O)[C@H](F)CNC(=O)c1cnc(Nc2ccn3nccc3c2)cc1NC1CCC(F)C1
InChIKey: HCYLDIXADIYEMN-NODKNVJMSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 6.2 nM (pChEMBL 8.210000038146973)
Descriptors: MW 458.51, ALogP 3.61, HBA 7, HBD 4, PSA 103.58, QED 0.41
Compound (SMILES): Cc1cc(C(F)(F)F)cc2[nH]c(Cc3c(Cl)ccc(C(=O)N4CCC(N(C)C)CC4)c3Cl)cc12
InChIKey: NHDQEJUGJPGBEB-UHFFFAOYSA-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 512.4, ALogP 6.56, HBA 2, HBD 1, PSA 39.34, QED 0.43
Compound (SMILES): c1ncc(-c2c[nH]c3nccc(O[C@@H]4CCCNC4)c23)cn1
InChIKey: LIZZRPJKAHJFGK-GFCCVEGCSA-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 = 4.01 nM (pChEMBL 8.399999618530273)
Descriptors: MW 295.35, ALogP 2.15, HBA 5, HBD 2, PSA 75.72, QED 0.77
Compound (SMILES): C=CC(=O)N1CCC(COc2ncnc(N)c2-c2ccc(Oc3ccccc3)cc2)C1
InChIKey: XEKQHZUWQALBJY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Enzyme Assay: The following describes a microfluidic, off-chip mobility shift kinase assay used to measure inherent potency of compounds against BTK enzyme. Compounds described by embodiments of the present invention were assayed using this protocol and the data from the same is recorded in Table 2 within the column labeled: Time Dependent BTK Enzyme Assay IC50. These IC50 values are reported in ranges wherein: A<100 nM, B<1 uM, and C>1 uM.2.5× stocks of full-length human BTK (08-080) from CarnaBio USA, Inc., Natick, Mass., 1.6×ATP and appropriate kinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2) were prepared in kinase reaction buffer consisting of 25 mM MgCl2, 0.015% Brij-35 (30%), 100 mM Hepes, pH 7.5, and 10 mM DTT.5 uL of enzyme buffer and 7.5 uL of ATP/kinKDR peptide substrate mix were added to Matrix (#115304) 384-well, sterile, polypropylene plates (Thermo Fisher Scientific, Hudson, N.H.) with 125 nL of serially diluted compounds prepared in 100% DMSO, and incubated for 90 min. at 27 C. Following the incubation period, reactions were stopped by adding 60 uL stop buffer consisting of 100 mM Hepes, pH 7.5, 0.015% Brij-35 (30%), 0.277% Coating Reagent #3 (Caliper Life Sciences, Mountain View, Calif.), 5% DMSO. Stopped reactions were monitored at −2 PSI, −3000 V/−700 V in a LabChip 3000 plate reader from Caliper Life Sciences, a PerkinElmer Company (Hopkinton, Mass.), and the activity was measured by off-chip mobility shift assay measuring the charge/mass difference between substrate and product resulting from peptide phosphorylation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. % Activity in GeneData Screener (Basel, Switzerland). Compounds described by embodiments of the present invention were assayed using this protocol and the data from the same is recorded in Table 2 within the column labeled: Time Dependent PBMC BTK Enzyme Assay IC50.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 416.48, ALogP 3.93, HBA 6, HBD 1, PSA 90.57, QED 0.59
Compound (SMILES): COc1ccnc(NCc2cc3cc(Cl)ccc3[nH]c2=O)c1
InChIKey: VNJKIUJTLMAMNW-UHFFFAOYSA-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 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 315.76, ALogP 3.2, HBA 4, HBD 2, PSA 67.01, QED 0.78
Compound (SMILES): CC(C)COc1cc(-c2ccc(N3CCN(Cc4ncccn4)CC3)nc2)c2c(C#N)cnn2c1
InChIKey: FIHZVQFJMGFNIW-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF® KinEASE¿-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 128 nM (pChEMBL 6.889999866485596)
Descriptors: MW 468.57, ALogP 3.41, HBA 9, HBD 0, PSA 95.47, QED 0.41
Compound (SMILES): O=C(Nc1nnc(-c2ccccc2F)s1)c1ccc(OC(F)(F)F)c(NC(=O)C2(N3CCOCC3)CC2)c1
InChIKey: UIRJNEQFJAASIK-UHFFFAOYSA-N
Target: Protein Wnt-3a (SINGLE PROTEIN), Homo sapiens
UniProt: P56704
Assay: HCT116 TOPFlash Assay: In preparation of the assay, the two cell lines were plated 24 hours before at 10000 cells per well of a 384 micro titre plate (MTP) in 30 μL growth medium. Selective inhibitory activity for small molecules on the mutated Wnt pathway was determined after parallel incubation of both (TOP and FOP) HCT116 reporter cell lines with a compound dilution series from 50 μM to 15 nM in steps of 3.16-fold dilutions in CAFTY buffer (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl2, 5 mM NaHCO3, pH 7.4) containing 2 mM Ca2+ and 0.01% BSA. The compounds were thereby serially prediluted in 100% DMSO and thereafter in addition 50 fold into the CAFTY compound dilution buffer (described above). From this dilution 10 μL were added to the cells in 30 μL growth medium and incubated for 36 hours at 37° C. and 5% CO2. Thereafter luciferase assay buffer (1:1 mixture of luciferase substrate buffer (20 mM Tricine, 2.67 mM MgSO4, 0.1 mM EDTA, 4 mM DTT, 270 μM Coenzyme A, 470 μM Luciferin, 530 μM ATP, ph adjusted to pH 7.8 with a sufficient volume of 5M NaOH) and Triton buffer (30 mL Triton X-100, 115 mL glycerol, 308 mg Dithiothreitol, 4.45 g Na2HPO4.2H2O, 3.03 g TRIS HCl, ad 1I H2O, pH 7.8) was added as equal volume to the compound solution on the cells to determine luciferase expression as a measure of Wnt signaling activity in a luminometer.n order to determine the inhibitory activity of compounds for the WT Wnt signaling pathway, the Super TopFlash vector were cotransfected with pcDNA3 into HEK293 cells.
Measurement: IC50 = 216 nM (pChEMBL 6.670000076293945)
Descriptors: MW 551.52, ALogP 4.3, HBA 8, HBD 2, PSA 105.68, QED 0.42
Compound (SMILES): C=C(C)c1cn(-c2cc(C)ccc2F)c(=O)c2cc(F)c(-n3nc(CO)n(CC)c3=O)cc12
InChIKey: XYJWHXGXSDQQPV-UHFFFAOYSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: In Vitro Assay: DHODH Enzymatic Assay: The following assay is referred to herein as the DHODH Enzymatic Assay. To detect DHODH enzyme activities, dichloroindophenol (DCIP) is added as the final electron acceptor in the assay. DCIP can accept electrons from the reduced coenzyme Q generated in the assay, or from dihydroorotate (DHO) via FMN by binding presumably to the ubiquinone pocket. DCIP solutions are blue, with an intense absorbance around 600 nm, but becomes colorless upon reduction (J. Biol. Chem. (1986) 261, 11386). The assay buffer contained 50 nM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, and 0.1% Triton X-100 in MilliQ water. Substrate consisting of 20 mM DHO, 5 mM CoQ6, and 1 mM DCIP in assay buffer, initiates the reaction. The assay is run in end-point mode by quenching the reaction with the potent DHODH inhibitor brequinar. Absorbance measurements were obtained using the BMG Phera Star plate-reading spectrophotomer. Purified human DHODH was purchased from Proteros (cat. No. PR-0044). Chemicals were purchased from Sigma-Aldrich, Teknova, and Avanti Polar Lipids. Liquid handling was performed using Labcyte Echo and Formulatrix Tempest.
Measurement: IC50 = 1.69 nM (pChEMBL 8.770000457763672)
Descriptors: MW 452.46, ALogP 3.47, HBA 7, HBD 1, PSA 82.05, QED 0.5
Compound (SMILES): CC1CCC23CCC4(C)C5(C)CCC6C(C)(C)C(=O)CCC6(C)C5C=CC4(OC2)C3C1C
InChIKey: PKMBOLUTQNQQBP-UHFFFAOYSA-N
Target: Tyrosine-protein phosphatase non-receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P18031
Assay: Metabolic Measurements: Glucose in tail blood was measured using a glucometer (One-Touch Basic; Lifescan, CA). For glucose tolerance tests (GTTs), mice were fasted for 10 hours and then injected with 20% D-glucose (2 mg/g body weight) and the blood glucose was monitored immediately before and at 15, 30, 60 and 120 mins following the injection. For insulin tolerance tests (ITTs), 4-h fasted animals were given insulin (0.75 mU/g) and blood glucose was measured immediately before and at 30, 60 and 120 minutes postinjection. Serum insulin, cholesterol, triglycerides (Stanbio Labs, TX), BDNF (Abnova), IGF1 and IGFBPs (R&D Systems) were determined by enzyme-linked immunosorbent assay.
Measurement: Ki = 13000 nM (pChEMBL 4.889999866485596)
Descriptors: MW 438.7, ALogP 7.22, HBA 2, HBD 0, PSA 26.3, QED 0.37
Compound (SMILES): CC(C)Cc1ncc(-c2cc(COC(C)(C)C(=O)O)nn2Cc2ccccc2Cl)o1
InChIKey: FLIRPDCLHDVEKB-UHFFFAOYSA-N
Target: Monocarboxylate transporter 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O15427
Assay: MCT4-Mediated Lactate Transport in MDA-MB-231 Breast Cancer Cells: MDA-MB-231 breast cancer cells may be used to measure MCT4 activity in cells with high native levels of MCT4 and low levels of MCT1 and are known to those with skill in the art. MCT4 activity may be assessed by monitoring the intracellular pH change that accompanies lactate/proton symport, using the pH-sensitive fluorescent dye 2′,7′-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), in a manner similar to that previously reported for MCT1 and MCT4. The following is an exemplary procedure for assaying MCT4 activity of the compounds of Formula (I).
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 431.92, ALogP 4.82, HBA 6, HBD 1, PSA 90.38, QED 0.52
Compound (SMILES): CC(=O)c1nn(CC(=O)N(CC(=O)Nc2cccc(-c3ccccc3Cl)c2F)C(C)C)c2ccc(C(=O)O)cc12
InChIKey: DIKNYSOOINWAQD-UHFFFAOYSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Inhibition Assay: An established esterolytic assay for the measurement of Factor D activity and inhibition of Factor D activity was used (Kam, C. M.; McRae, B. J.; Harper, J. W.; Niemann, M. A.; Volanakis, J. E.; Powers, J. C. Human complement proteins D, C2, and B Active site mapping with peptide thioester substrates. J Biol. Chem. 1987, 262, 3444-3451). For this assay Z-Lys-SBzl, 1.29 mM (Kim, S.; Narayana, S. V. L; Volanakis, J. E. Mutational analysis of the substrate binding site of human complement Factor D. Biochemistry. 1994, 33, 14393-14399) was used as the substrate for Factor D (104 mM). Hydrolysis of this compound by Factor D liberated a free sulfhydryl group which is then reacted with 5,5′-dithiobis(2nitrobenzoic acid) producing an intense yellow color (Habeeb, A. F. S. A. Reaction of protein sulfhydryl groups with Ellman's Reagent. Methods in Enzymol. 1976, 25, 457-464.). The assays were performed in 96 well microtiter plates and rates of hydrolysis were monitored at 405 nm on a Biotek Synergy H1 plate reader. Hydrolysis rates were reported as change in mOD/min. The assay was conducted in 100 mM HEPES, 500 mM NaCl, pH 7.5 containing 10% DMSO in a final volume of 50 μL per well.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 565, ALogP 5.27, HBA 6, HBD 2, PSA 121.6, QED 0.27
Compound (SMILES): Cc1noc(C)c1Cn1c(=O)[nH]c2ncc(-c3cccc(C#N)c3)cc21
InChIKey: POFFFDGIBCRIQW-UHFFFAOYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: HTS cell-based calcium flux assay: 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 (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 > 29000 nM (pChEMBL 4.538000106811523)
Descriptors: MW 345.36, ALogP 2.92, HBA 6, HBD 1, PSA 100.5, QED 0.62
Compound (SMILES): Cc1nc(-c2ccc(F)cc2)c(-c2ccc3nc(NC(=O)C4CCC(=O)N4)cn3n2)n1C
InChIKey: VKAYXDAUXCCSPJ-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 25.1 nM (pChEMBL 7.599999904632568)
Descriptors: MW 433.45, ALogP 2.46, HBA 7, HBD 2, PSA 106.21, QED 0.51
Compound (SMILES): Cc1cc(-c2n[nH]c3cc4c(cc23)CN(C2CN(CC3CC3)C2)C(=O)N4)ccn1
InChIKey: MGLHYQCTSHZWGH-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Z′-LYTE Kinase Assay: The inhibition of ERK activity by the compounds disclosed herein was determined using the Z′-LYTE kinase assay kit (Life Technologies) with a Ser/Thr 3 peptide substrate (Life Technologies) according to manufacturers' instructions. The assay was run with an ERK2 enzyme (Life Technologies) concentration of 0.47 ng/μL at 100 μM ATP (approximately the ATP Km for ERK2). The IC50 values for the compounds were determined with 3-fold serial dilutions in duplicate. The compounds were first diluted in 1:3 dilutions in 100% DMSO at 100× the desired concentration, and then further diluted (1:25) in 20 mM HEPES buffer (Invitrogen) to make 4× solutions prior to adding to the enzyme solution. The final DMSO concentration in the assay was 1%. Final reaction volume was 20 μL/well in 384-well plates. Kinase reactions were conducted or 1 hour followed by the assay development reaction (1 hour) in a 20 ul/well in a 384 well plate format.
Measurement: IC50 = 625 nM (pChEMBL 6.199999809265137)
Descriptors: MW 388.48, ALogP 3.38, HBA 4, HBD 2, PSA 77.15, QED 0.72
Compound (SMILES): COc1cc(OCc2cccc(NC(=O)c3csc(-c4ccccc4)n3)c2)c2cc(-c3cn4nc(OC)sc4n3)oc2c1
InChIKey: XWRGTAFUIGYUPW-UHFFFAOYSA-N
Target: Proteinase-activated receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI0
Assay: FLIPR Assay in PAR4-Expressing HEK293 Cells: FLIPR-based calcium mobilization assay in HEK293 cells was used to measure PAR4 antagonism, agonism, and selectivity against PAR1. The activity of the PAR4 antagonists of the present invention were tested in PAR4 expressing cells by monitoring H-Ala-Phe(4-F)-Pro-Gly-Trp-Leu-Val-Lys-Asn-Gly-NH2-induced intracellular calcium mobilization. Counter screens for agonist activity and PAR1 antagonist activity were also performed. Briefly, PAR1/PAR4-expressing HEK293 cells were grown in DMEM (Life Technology, Grand Island, N.Y.) containing 10% heat-inactivated FBS, 1% Penicillin-Streptomycin, 10 μg/mL blasticidin, and 100 μg/mL Zeocin at 37° C. with 5% CO2. Cells were plated overnight prior to the experiment in a black 384-well Purecoat Amine clear bottom plate (Becton Dickinson Biosciences, San Jose, Calif.) at 10,000 cells/well in 30 μL growth medium and incubated in a humidified chamber at 37° C. with 5% CO2 overnight. Prior to compound addition, the cell medium was replaced with 40 L of 1× calcium and magnesium-containing Hank's Balanced Saline Solution (HBSS) (with 20 mM HEPES) and 1:1000 diluted fluorescent calcium indicator (Codex Biosolutions, Gaithersburg, Md.). After a 30 minute incubation period at 37° C. and a further 30 minute incubation and equilibration period at room temperature, 20 μL test compound (diluted in 1× HBSS buffer) was added at various concentrations at 0.17% dimethyl sulfoxide (DMSO) final concentration. Changes in fluorescence intensity were measured using a Functional Drug Screening System (FDSS, Hamamatsu, Japan) to determine agonist activities. The cells were then incubated for 30 minutes at room temperature followed by addition of 20 μL of agonist peptide for antagonist activity measurement. The PAR4 agonist peptide (H-Ala-Phe(4-F)-Pro-Gly-Trp-Leu-Val-Lys-Asn-Gly-NH2) and the PAR1 agonist peptide (SFFLRR) were routinely tested to ensure a proper response at the EC50 value in the assay (5 μM for PAR4 agonist peptide and 2 μM for PAR1 agonist peptide). Compound potency was derived from 11-point concentration-response curves.
Measurement: EC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 609.69, ALogP 7.18, HBA 11, HBD 1, PSA 113.01, QED 0.18
Compound (SMILES): CNC(=O)c1ccc2c(c1)NC(=O)c1csc(n1)-c1ccnc(c1)NCCCCCO2
InChIKey: XPKAFGUAGHEAEX-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, 10 μM 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 437.52, ALogP 3.79, HBA 7, HBD 3, PSA 105.24, QED 0.53
Compound (SMILES): Cc1ccc(-c2cn(CC(=O)N3CCC[C@@H](N)C3)nc2-c2ccc(C#N)c(F)c2)cc1
InChIKey: QDEYDJJAOOMSPG-HXUWFJFHSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Evaluation of LSD1 Enzyme Activity: The purpose of this assay was to detect the inhibitory activity of the compounds against LSD1 in vitro. The enzyme used in this assay was human LSD1 and the standard substrate was histone H3K4me peptide (20 M); the activity of the compounds was determined by the enzyme fluorescence coupling method using a combination of horseradish peroxidase (HRP) and Amplex Red to detect the H2O2 generated after the reaction. The IC50 values of the compounds were measured at 10 concentrations after 3 times dilution from 10 μm. Before the compound was added to the substrate to start the reaction, enzyme and substrate were incubated for 30 minutes. Fluorescence detector: EnVision, excitation wavelength: Ex/Em=530/590 nM.
Measurement: IC50 = 275 nM (pChEMBL 6.559999942779541)
Descriptors: MW 417.49, ALogP 3.49, HBA 5, HBD 1, PSA 87.94, QED 0.7
Compound (SMILES): CCCNC(=O)O[C@H]1CC[C@@H](c2cc(NC(=O)Cc3cc(OC)ncc3O)n[nH]2)C1
InChIKey: AVPYRRXTDTYCKW-OCCSQVGLSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: Biochemical Assay: The purpose of CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 full length catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2, CPC Scientific, Sunnyvale, Calif.) (SEQ ID NO:1). 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. Wild-type CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Matl enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. 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 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA.
Measurement: Ki = 0.41 nM (pChEMBL 9.390000343322754)
Descriptors: MW 417.47, ALogP 2.47, HBA 7, HBD 4, PSA 138.46, QED 0.52
Compound (SMILES): COC/C(C)=C1\SC(N2CCC3(CC2)OCc2ccccc23)=NC1=O
InChIKey: RZEYUUYQLROXHS-SSZFMOIBSA-N
Target: Vasopressin V1a receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P37288
Assay: competition binding assay: The human V1a receptor was cloned by RT-PCR from total human liver RNA. The coding sequence was subcloned in an expression vector after sequencing to confirm the identity of the amplified sequence. To demonstrate the affinity of the compounds from the present invention to the human V1a receptor binding studies were performed. Cell membranes were prepared from HEK293 cells transiently transfected with the expression vector and grown in 20 liter fermenters with the following protocol.50 g of cells are re-suspended in 30 ml freshly prepared ice cold Lysis buffer (50 mM HEPES, 1 mM EDTA, 10 mM MgCl2 adjusted to pH=7.4+complete cocktail of protease inhibitor (Roche Diagnostics)). Homogenized with Polytron for 1 min and sonicated on ice for 2×2 minutes at 80% intensity (Vibracell sonicator). The preparation is centrifuged 20 min at 500 g at 4° C., the pellet is discarded and the supernatant centrifuged 1 hour at 43,000 g at 4° C. (19,000 rpm). The pellet is re-suspended in 12.5 ml Lysis buffer+12.5 ml Sucrose 20% and homogenized using a Polytron for 1-2 min. The protein concentration is determined by the Bradford method and aliquots are stored at −80° C. until use. For binding studies 60 mg Yttrium silicate SPA beads (Amersham) are mixed with an aliquot of membrane in binding buffer (50 mM Tris, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 10 mM MgCl2) for 15 minutes with mixing. 50 μl of bead/membrane mixture is then added to each well of a 96 well plate, followed by 50 μl of 4 nM 3H-Vasopressin (American Radiolabeled Chemicals). For total binding measurement 100 μl of binding buffer are added to the respective wells, for non-specific binding 100 μl of 8.4 mM cold vasopressin and for compound testing 100 μl of a serial dilution of each compound in 2% DMSO. The plate is incubated 1 h at room temperature, centrifuged 1 min at 1000 g and counted on a Packard Top-Count. Non-specific binding counts are subtracted from each well and data is normalized to the maximum specific binding set at 100%. To calculate an IC 50 the curve is fitted using a non-linear regression model (XLfit) and the Ki is calculated using the Cheng-Prussoff equation.
Measurement: Ki = 3.98 nM (pChEMBL 8.399999618530273)
Descriptors: MW 358.46, ALogP 3.06, HBA 5, HBD 0, PSA 51.13, QED 0.76
Compound (SMILES): NCCCC[C@@H](NC1CCC2(CC1)CCN(Cc1ccccc1)C2)C(=O)N1CCN(C(=O)Oc2ccc3ccccc3c2Cl)C[C@H]1C(=O)NCc1cccs1
InChIKey: KHIWWPFSIJVVQL-QTUSZSIFSA-N
Target: Coagulation factor XII (SINGLE PROTEIN), Homo sapiens
UniProt: P00748
Assay: Factor XIIa Assay: The effectiveness of a compound of the present invention as an inhibitor of Coagulation Factor XIIa can be determined using a relevant purified serine protease, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Assays were conducted at room temperature or at 37° C. Hydrolysis of the substrate resulted in release of amino trifluoromethylcoumarin (AFC), which was monitored spectrofluorometrically by measuring the increase in emission at 510 nm with excitation at 405 nm. A decrease in the rate of fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the IC50, the inhibitor concentration causing a 50% decrease in Factor XIIa protease activity.Factor XIIa activity determinations were made in 50 mM HEPES buffer containing 150 mM NaCl, 5 mM CaCl2, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific) at pH 7.4. Determinations were made using purified human Factor XIIa at a final concentration of 500 pM (Sekisui Diagnostics) and the synthetic substrate, n-Acetyl-Lys-Pro-Arg-AFC, TFA salt (Sigma #C6608) at a concentration of 100 μM.Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration ≤0.1 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. Initial velocities of product formation in the absence (Vo) or presence of inhibitor (Vi) were measured. IC50 was determined as the concentration of I yielding Vi=Vo/2.
Measurement: IC50 = 22.7 nM (pChEMBL 7.639999866485596)
Descriptors: MW 785.46, ALogP 6.8, HBA 8, HBD 3, PSA 120.24, QED 0.13
Compound (SMILES): CC1Oc2cc(C(=O)NCc3cccc(C(=O)NCC4CC4)c3)ccc2-c2ccncc21
InChIKey: FJXFWZUOMXSEEO-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 8.97 nM (pChEMBL 8.050000190734863)
Descriptors: MW 427.5, ALogP 4.27, HBA 4, HBD 2, PSA 80.32, QED 0.62
Compound (SMILES): CCC(CO)n1cnc(-c2ccc(F)cc2)c1-c1ccc2ncc(C(N)=O)n2c1
InChIKey: FOWZNNSTDFRSKJ-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assay: Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule prode (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 393.42, ALogP 3.05, HBA 6, HBD 2, PSA 98.44, QED 0.53
Compound (SMILES): O=C1NCCCCc2ncc(F)cc2[C@H]2CCCN2c2ccn3ncc1c3n2
InChIKey: COFVTKKTPFIGHL-QGZVFWFLSA-N
Target: Proto-oncogene tyrosine-protein kinase ROS (SINGLE PROTEIN), Homo sapiens
UniProt: P08922
Assay: Inhibition Kinase Assay: The potency of a compound inhibiting wild type and exemplary mutant ROS1 kinases was determined using CisBio's HTRF Kinease-TK assay technology. The assays contained 5 nM wild type ROS1 (SignalChemCat. No. R14-11G), 5 nM G2032R ROS1 (SignalChemCat. No. R14-12BG), 5 nM L2026M ROS1 (Array Biopharma, p 1965), or 5 nM D2033N ROS1 (Array Biopharma, p 1994). Each kinase is incubated with 250 nM TK-substrate biotin (CisBio, Cat. No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM MOPS [pH 7.4], 5 mM MgCl2, 0.005% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were prepared in a four-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 120-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.3 nM Sa-XL665 and 1×TK-Ab-Cryptate in HTRF detection buffer (CisBio, Cat. No. 62TK0PEC).
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 380.43, ALogP 2.67, HBA 6, HBD 1, PSA 75.42, QED 0.65
Compound (SMILES): Cc1nnn(C)c1-c1cnc2c3c(N4CCCS4(=O)=O)ccc(S(C)(=O)=O)c3n([C@H](c3ccc(F)cc3)C3CCOCC3)c2c1
InChIKey: ZDBJQVVWZCAREA-SSEXGKCCSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The binding of compounds to bromodomain BRD4 (44-168), BRD4 (333-460), and BRD4 (1-477 or 44-460) was assessed using a time resolved fluorescent resonance energy transfer binding assay (1), that measures the binding of a fluorescently labeled probe molecule to the bromodomain protein. The bromodomain protein, fluorescent probe molecule (either a biotinylated histone peptide or a fluorescently labeled small molecule), and dose-responsed test compound are incubated together to reach thermodynamic equilibrium. In the absence of a test compound, the bromodomain and small molecule are bound, resulting in a high fluorescent signal. In the presence of a sufficient concentration of inhibitor, this interaction is disrupted resulting in a lost of fluorescent resonance energy transfer.All assay components were dissolved in buffer composition 20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM DTT, 0.005% Tween 20, and 100 ug/ml BSA for BRD4 (1-477 and 44-460). The final concentrations of the bromodomain proteins are 1.6 nM BRD4(44-168), 1 nM BRD4(333-460), and 1 nM BRD4(1-477 or 44-460), and the fluorescent probe molecule is 100 nM, 50 nM, and 7.5 nM respectively. All proteins were biotinylated. A streptavidin labeled with terbium cryptate (Cisbio SA-Tb) was used as detection, and pre-mixed with the bromodomain protein at a final concentration of 0.2 nM. In some instances for BRD4 (44-460), anti-His terbium cryptate was used as a detection. 7.5 nl of dose-responsed test compound or dmso vehicle (0.0375%) was pre-spotted in a black Corning 384 well plate and 10 ul each of bromodomain/detection reagent and fluorescent small molecule solution were added to the plate, and the reaction incubated for 60 min at room temperature. Plates were then read on EnVision plate reader, (λex=340 nm, acceptor λEm=520 nm, and donor λEm=615 nm, LANCE D400 mirror). Time resolved fluorescence intensity measurements were made at both emissions, and the ratio of acceptor/donor was calculated and used for data analysis. All data was normalized to 16 high vehicle wells and 8 low reference control wells, and then a four parameter curve fit was applied:Y=a+((b−a)/(1+(10x/10c)d)Where 'a' is the minimum, 'b' is the Hill slope, 'c' is the IC50, and 'd' is the maximum.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 652.77, ALogP 4.39, HBA 10, HBD 0, PSA 129.28, QED 0.26
Compound (SMILES): Cc1nn(-c2cccnc2F)c2c1C(=O)N(c1cc(C)c(=O)n(C)c1)C2c1ccc(Cl)cc1
InChIKey: STEDHESXTGJFOX-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET In-Vitro Binding Assays for BRD2, BRD3, and BRD4: All assays were performed in 384 well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Perkin Elmer/Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO HummingBird nanodispenser (Zinsser Analytic). The assay was started by stepwise addition of 4.5 μl per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 45 nM His-Brd2(60-472) or 45 nM His-Brd3(20-477) or 45 nM His-Brd4(44-477) all proteins produced in-house) and 4.5 μl per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 60 nM acetyl-histone H4 (AcK 5, 8, 12, 16) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 μl per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 3 nM Eu-labeled anti-His6 antibody, 21 nM streptavidin-allophycocyanin) were added. After 35 minutes incubation at 30° C., plates were measured in a Perkin Elmer EnVision multilabel reader. Concentrations causing 50% inhibition (1050) values were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 463.9, ALogP 4.13, HBA 6, HBD 0, PSA 73.02, QED 0.43
Compound (SMILES): CCS(=O)(=O)N(C)c1cncc(-c2ccc3nc(-c4nc5ccccc5[nH]4)oc3c2)c1
InChIKey: CDIOSALLCLKISA-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Scintillation Proximity Assay (PI3K-γ): [γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (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). PI3Kγ (p110γ) Recombinant Human Protein was purchased from Life technology (Grand Island, N.Y.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one 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 2%. The PI3Kγ assay was 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, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer).
Measurement: IC50 < 250 nM (pChEMBL 6.6020002365112305)
Descriptors: MW 433.49, ALogP 4.22, HBA 6, HBD 1, PSA 104.98, QED 0.44